Biomass compositions for decreasing bruising in fruit and methods therefor

ABSTRACT

Biomass compositions and methods for decreasing bruising of fruit of a fruiting plant by administering to the fruiting plant, seedling, or seed, a liquid composition treatment comprising a culture of microalgae cells are disclosed. The liquid composition may comprise pasteurized  Chlorella  cells only,  Aurantiochytrium acetophilum  HS399 cells only, or a combination of  Chlorella  and  Aurantiochytrium acetophilum  HS399 cells that are pasteurized at a temperature between 65° C.-90° C. The administration may comprise contacting soil in the immediate vicinity of the fruiting plant, seedling, or seed.

CROSS-REFERENCE TO RELATED APPLICATIONS

This application claims the benefit of Provisional Application No.62/584,311 entitled “BIOMASS COMPOSITIONS,” which was filed on Nov. 10,2017 in the name of the Applicant and which is incorporated herein infull by reference. This application also claims the benefit ofProvisional Application No. 62/680,373 entitled “BIOMASS COMPOSITIONS,”which was filed on Jun. 4, 2018 in the name of the Applicant and whichis incorporated herein in full by reference.

FIELD OF THE INVENTION

The present invention generally relates to agriculture and, morespecifically, to biomass compositions and methods for decreasingbruising in fruits of fruiting plants.

BACKGROUND OF THE INVENTION

The growth of a plant is a complex physiological process involvinginputs and pathways in the roots, shoots, and leaves. Whether at acommercial or home garden scale, growers are constantly striving tooptimize the yield and quality of plants, which may include decreasingthe amount of bruising in fruits from fruiting plants.

SUMMARY OF THE INVENTION

This Summary is provided to introduce a selection of concepts in asimplified form that are further described below in the DetailedDescription. This Summary is not intended to identify key factors oressential features of the claimed subject matter, nor is it intended tobe used to limit the scope of the claimed subject matter.

Embodiments of the invention relate to a composition for enhancing atleast one plant characteristic. The composition can include a microalgaebiomass that includes at least one species of microalgae. Thecomposition can include a microalgae biomass that includes at least twospecies of microalgae. The composition can cause synergistic enhancementof at least one plant characteristic.

In some embodiments, the microalgae species can include Chlorella,Schizochytrium, Thraustochytrium, Oblongichytrium and/orAurantiochytrium acetophilum HS399. In other embodiments, the microalgaespecies can include Botryococcus, Chlamydomonas, Scenedesmus, Pavlova,Phaeodactylum, Nannochloropsis, Spirlulina, Galdieria, Haematococcus,Isochrysis, Porphyridium, Tetraselmis, and/or the like.

In some embodiments, the microalgae biomass can include whole biomassand/or residual biomass. Whole biomass includes substantially allcomponents and fractions of the cells from which the whole biomass isderived. Residual or extracted biomass can be any remaining biomassafter extraction and/or removal of one or more components of a wholebiomass.

In some embodiments, the composition can include one species ofmicroalgae. In some embodiments, the composition can include a firstspecies of microalgae and a second species of microalgae. The ratio ofthe first species of microalgae and the second species of microalgae canbe between about 25:75, 50:50, or 75:25.

In some embodiments, the first species of microalgae may be Chlorellaand the second species of microalgae may be Aurantiochytrium acetophilumHS399. In some embodiments, the ratio of Chlorella and Aurantiochytriumacetophilum HS399 may range between about 25:75 to 75:25. For example,the ratio of Chlorella and Aurantiochytrium acetophilum HS399 may beabout 25:75, 50:50, or 75:25. In some embodiments, the Chlorella iswhole biomass and Aurantiochytrium acetophilum HS399 isresidual/extracted biomass. In some embodiments, the Aurantiochytriumacetophilum HS399 is whole biomass and Chlorella is residual/extractedbiomass. In some embodiments, the Chlorella and Aurantiochytriumacetophilum HS399 are both whole biomass and in other embodiments theChlorella and Aurantiochytrium acetophilum HS399 are bothresidual/extracted biomass.

Some embodiments of the invention relate to a method of plantenhancement comprising administering to a plant, seedling, seed, or soilthe composition treatment, wherein the composition treatment enhances atleast one plant characteristic. In some embodiments, the composition isapplied when the plant is under salt stress conditions, temperaturestress conditions, and/or the like.

Embodiments of the invention relate to a method of plant enhancementcomprising administering a composition treatment comprising at least onemicroalgae species to soil. The administering can be by soil drench atthe time of seeding. The method can include growing the plant to atransplant stage. The method can include transferring the plant at thetransplant stage from an initial container to a larger container or afield, or the like. In some embodiments the plant at the transplantstage has at least one enhanced plant characteristic. The enhanced plantcharacteristic can be improved root density, improved root area,enhanced plant vigor, enhanced plant growth rate, enhanced plantmaturation, and/or enhanced shoot development. After the transfer, theplant may have at least one enhanced plant characteristic. Thecomposition treatment can include at least one microalgae species suchas Botryococcus, Chlorella, Chlamydomonas, Scenedesmus, Pavlova,Phaeodactylum, Nannochloropsis, Aurantiochytrium, Spirlulina, Galdieria,Haematococcus, Isochrysis, Porphyridium, Schizochytrium, Tetraselmis,and/or the like.

In some of the embodiments and Examples below, the microalgaecomposition may be applied to the soil of the fruiting plant bydrenching the soil initially at the time of transplant and thensubsequently every two weeks (once every 14 days) after transplant untilharvest.

In one embodiment of the present invention, a method of decreasingbruising of fruit of a fruiting plant is disclosed. The method comprisesthe step of administering to the fruiting plant, seedling, or seed aliquid composition treatment comprising a culture of microalgae, themicroalgae comprising at least one of pasteurized Chlorella cells andpasteurized Aurantiochytrium acetophilum HS399 cells in an amounteffective to decrease bruising in the fruit of a population of suchfruiting plants compared to a substantially identical population ofuntreated fruiting plants.

In another embodiment of the present invention, a method of decreasingbruising of fruit of a fruiting plant is disclosed. The method comprisesthe step of administering to the fruiting plant, seedling, or seed aliquid composition treatment comprising a culture of microalgae, themicroalgae comprising at least one of pasteurized Chlorella cells andpasteurized Aurantiochytrium acetophilum HS399 cells in an effectiveamount to decrease bruising in the fruit of a population of fruitingplants by 2-100% compared to fruit of a substantially identicalpopulation of untreated fruiting plants, wherein administeringcomprising contacting soil in the immediate vicinity of the fruitingplant, seedling, or seed with an effective amount of the liquidcomposition treatment by drip irrigation.

In another embodiment of the present invention, a method of decreasingbruising of fruit of a fruiting plant is disclosed. The method comprisesthe steps of: providing a liquid composition treatment comprising aculture of microalgae, the microalgae comprising at least one ofpasteurized Chlorella cells and pasteurized Aurantiochytrium acetophilumHS399 cells; diluting the liquid composition treatment to containbetween 100 g-800 g per acre of the at least one of pasteurizedChlorella cells and pasteurized Aurantiochytrium acetophilum HS399cells; and administering the liquid composition treatment to a fruitingplant, seedling, or seed in an effective amount to decrease bruising inthe fruit of a population of such fruiting plants compared to asubstantially identical population of untreated fruiting plants.

To the accomplishment of the foregoing and related ends, the followingdescription and annexed drawings set forth certain illustrative aspectsand implementations. These are indicative of but a few of the variousways in which one or more aspects may be employed. Other aspects,advantages and novel features of the disclosure will become apparentfrom the following detailed description when considered in conjunctionwith the annexed drawings.

BRIEF DESCRIPTION OF THE DRAWINGS

The present application is further detailed with respect to thefollowing drawings. These figures are not intended to limit the scope ofthe present application, but rather, illustrate certain attributesthereof.

FIG. 1 is a table showing the effects of several microalgae compositionson strawberry quality after storage, wherein the effects are observed ina decrease in bruising and increases in appearance, aroma, flavor, andtexture relative to the UTC;

FIG. 2 is a graph showing the effects of one of the microalgaecompositions of FIG. 1 on strawberry quality after storage, wherein theeffects are observed in an increase in post-storage marketabilityrelative to the UTC;

FIG. 3 is a graph showing the effects of one of the microalgaecompositions of FIG. 1 on strawberry quality after storage, wherein theeffects are observed in an increase in consumer preference based onappearance, aroma, flavor, and texture relative to the UTC;

FIG. 4 is a graph showing a comparison of the effects of severalmicroalgae compositions on strawberry shelf-life and post-harvestquality, wherein the effects are observed in an increase in post-harvestmarketability relative to the UTC and a seaweed commercial referenceproduct;

FIG. 5 is a graph showing a comparison of the effects of the severalmicroalgae compositions of FIG. 4 on strawberry shelf-life andpost-harvest quality, wherein the effects are observed in a decrease insevere and moderate bruising of the strawberries relative to the UTC anda seaweed commercial reference product;

FIG. 6 is a graph showing a comparison of the effects of the severalmicroalgae compositions of FIG. 4 on strawberry shelf-life andpost-harvest quality, wherein the effects are observed in an increase inconsumer preference based on appearance, aroma, flavor, and texturerelative to the UTC and a seaweed commercial reference product;

FIG. 7 is a graph showing a comparison of the effects of severalmicroalgae compositions on strawberry growth, yield, and post-harvestquality, wherein the effects are observed in a decrease in bruising ofthe strawberries after 5 days relative to the UTC and a seaweedcommercial reference product;

FIG. 8 is a graph showing a comparison of the effects of the severalmicroalgae compositions of FIG. 7 on strawberry growth, yield, andpost-harvest quality, wherein the effects are observed in a decrease inbruising of the strawberries after 7 days relative to the UTC and aseaweed commercial reference product;

FIG. 9 is a table showing a comparison of the effects of severalmicroalgae compositions on strawberry growth, yield, and post-harvestquality, wherein the effects are observed in a decrease in bruising, adecrease in fruit water loss, and an increase in berry firmness relativeto the UTC and a microbial-based commercial reference product;

FIG. 10 is a graph showing a comparison of the effects of the severalmicroalgae compositions of FIG. 9 on strawberry growth, yield, andpost-harvest quality, wherein the effects are observed in an increase inmarketability based on bruising relative to the UTC and amicrobial-based commercial reference product;

FIG. 11 is a graph showing a comparison of the effects of the severalmicroalgae compositions of FIG. 9 on strawberry growth, yield, andpost-harvest quality, wherein the effects are observed in a decrease inbruising relative to the UTC and a microbial-based commercial referenceproduct;

FIG. 12 is a graph showing a comparison of the effects of the severalmicroalgae compositions of FIG. 9 on strawberry growth, yield, andpost-harvest quality, wherein the effects are observed in an increase inconsumer preference based on appearance, aroma, flavor, and texturerelative to the UTC and a microbial-based commercial reference product;

FIG. 13 is a table showing a comparison of the effects of severalmicroalgae compositions on strawberry growth, yield, and post-harvestquality, wherein the effects are observed in a decrease in bruisingrelative to the UTC and a seaweed commercial reference product;

FIG. 14 is a continuation of the table shown in FIG. 13;

FIG. 15 is a graph showing a comparison of the effects of the severalmicroalgae compositions of FIG. 13 on strawberry growth, yield, andpost-harvest quality, wherein the effects are observed in an increase inmarketability relative to the UTC and a seaweed commercial referenceproduct;

FIG. 16 is a graph showing a comparison of the effects of the severalmicroalgae compositions of FIG. 13 on strawberry growth, yield, andpost-harvest quality, wherein the effects are observed in a decrease inbruising relative to the UTC and a seaweed commercial reference product;and

FIG. 17 is a graph showing a comparison of the effects of the severalmicroalgae compositions of FIG. 13 on strawberry growth, yield, andpost-harvest quality, wherein the effects are observed in an increase inconsumer preference based on appearance, aroma, flavor, and texturerelative to the UTC and a seaweed commercial reference product.

DETAILED DESCRIPTION OF THE INVENTION

The description set forth below in connection with the appended drawingsis intended as a description of presently preferred embodiments of thedisclosure and is not intended to represent the only forms in which thepresent disclosure may be constructed and/or utilized. The descriptionsets forth the functions and the sequence of steps for constructing andoperating the disclosure in connection with the illustrated embodiments.It is to be understood, however, that the same or equivalent functionsand sequences may be accomplished by different embodiments that are alsointended to be encompassed within the spirit and scope of thisdisclosure.

Many plants can benefit from the application of liquid compositions thatprovide a bio-stimulatory effect. Non-limiting examples of plantfamilies that can benefit from such compositions include plants from thefollowing: Solanaceae, Fabaceae (Leguminosae), Poaceae, Roasaceae,Vitaceae, Brassicaeae (Cruciferae), Caricaceae, Malvaceae, Sapindaceae,Anacardiaceae, Rutaceae, Moraceae, Convolvulaceae, Lamiaceae,Verbenaceae, Pedaliaceae, Asteraceae (Compositae), Apiaceae(Umbelliferae), Araliaceae, Oleaceae, Ericaceae, Actinidaceae,Cactaceae, Chenopodiaceae, Polygonaceae, Theaceae, Lecythidaceae,Rubiaceae, Papveraceae, Illiciaceae Grossulariaceae, Myrtaceae,Juglandaceae, Bertulaceae, Cucurbitaceae, Asparagaceae (Liliaceae),Alliaceae (Liliceae), Bromeliaceae, Zingieraceae, Muscaceae, Areaceae,Dioscoreaceae, Myristicaceae, Annonaceae, Euphorbiaceae, Lauraceae,Piperaceae, Proteaceae, and Cannabaceae.

The Solanaceae plant family includes a large number of agriculturalcrops, medicinal plants, spices, and ornamentals in its over 2,500species. Taxonomically classified in the Plantae kingdom, Tracheobionta(subkingdom), Spermatophyta (superdivision), Magnoliophyta (division),Manoliopsida (class), Asteridae (subclass), and Solanales (order), theSolanaceae family includes, but is not limited to, potatoes, tomatoes,eggplants, various peppers, tobacco, and petunias. Plants in theSolanaceae can be found on all the continents, excluding Antarctica, andthus have a widespread importance in agriculture across the globe.

The Rosaceae plant family includes flowering plants, herbs, shrubs, andtrees. Taxonomically classified in the Plantae kingdom, Tracheobionta(subkingdom), Spermatophyta (superdivision), Magnoliophyta (division),Magnoliopsida (class), Rosidae (subclass), and Rosales (order), theRosaceae family includes, but is not limited to, almond, apple, apricot,blackberry, cherry, nectarine, peach, plum, raspberry, strawberry, andquince.

The Fabaceae plant family (also known as the Leguminosae) comprises thethird largest plant family with over 18,000 species, including a numberof important agricultural and food plants. Taxonomically classified inthe Plantae kingdom, Tracheobionta (subkingdom), Spermatophyta(superdivision), Magnoliophyta (division), Manoliopsida (class), Rosidae(subclass), and Fabales (order), the Fabaceae family includes, but isnot limited to, soybeans, beans, green beans, peas, chickpeas, alfalfa,peanuts, sweet peas, carob, and liquorice. Plants in the Fabaceae familycan range in size and type, including but not limited to, trees, smallannual herbs, shrubs, and vines, and typically develop legumes. Plantsin the Fabaceae family can be found on all the continents, excludingAntarctica, and thus have a widespread importance in agriculture acrossthe globe. Besides food, plants in the Fabaceae family can be used toproduce natural gums, dyes, and ornamentals.

The Poaceae plant family supplies food, building materials, andfeedstock for fuel processing. Taxonomically classified in the Plantaekingdom, Tracheobionta (subkingdom), Spermatophyta (superdivision),Magnoliophyta (division), Liliopsida (class), Commelinidae (subclass),and Cyperales (order), the Poaceae family includes, but is not limitedto, flowering plants, grasses, and cereal crops such as barely, corn,lemongrass, millet, oat, rye, rice, wheat, sugarcane, and sorghum. Typesof turf grass found in Arizona include, but are not limited to, hybridBermuda grasses (e.g., 328 tifgrn, 419 tifway, tif sport).

The Vitaceae plant family includes flowering plants and vines.Taxonomically classified in the Plantae kingdom, Tracheobionta(subkingdom), Spermatophyta (superdivision), Magnoliophyta (division),Magnoliopsida (class), Rosidae (subclass), and Rhammales (order), theVitaceae family includes, but is not limited to, grapes.

Particularly important in the production of fruit from plants is thebeginning stage of growth where the plant emerges and matures intoestablishment. A method of treating a seed, seedling, or plant todirectly improve the germination, emergence, and maturation of theplant; or to indirectly enhance the microbial soil community surroundingthe seed or seedling is therefore valuable starting the plant on thepath to marketable production. The standard typically used for assessingemergence is the achievement of the hypocotyl stage, where a stem isvisibly protruding from the soil. The standard typically used forassessing maturation is the achievement of the cotyledon stage, wheretwo leaves visibly form on the emerged stem. Some botanists view thebeginning of maturation as starting at when the first true leaf emergesbeyond the cotyledon stage, as the cotyledons are already pre-formed inthe seed prior to germination. Some botanists see maturation as a longphase that proceeds until full reproductive potential has been achieved.

Important in the production of fruit from plants is the yield andquality of fruit, which can be quantified as the number, weight, color,firmness, ripeness, sweetness, moisture, degree of insect infestation,degree of disease or rot, degree of sunburn of the fruit. A method oftreating a plant to directly improve the characteristics of the plant,or to indirectly enhance the chlorophyll level of the plant forphotosynthetic capabilities and health of the plant's leaves, roots, andshoot to enable robust production of fruit is therefore valuable inincreasing the efficiency of marketable production. Marketable andunmarketable designations can apply to both the plant and fruit, and canbe defined differently based on the end use of the product, such as butnot limited to, fresh market produce and processing for inclusion as aningredient in a composition. The marketable determination can assesssuch qualities as, but not limited to, color, insect damage, blossom endrot, softness, and sunburn. The term “total production” can incorporateboth marketable and unmarketable plants and fruit. The ratio ofmarketable plants or fruit to unmarketable plants or fruit can bereferred to as “utilization” and expressed as a percentage. Theutilization can be used as an indicator of the efficiency of theagricultural process as it shows the successful production of marketableplants or fruit, which will be obtain the highest financial return forthe grower, whereas total production will not provide such anindication.

To achieve such improvements in emergence, maturation, and yield ofplants, a method to treat such seeds and plants, and soil with alow-concentration microalgae-based composition, in a dried or liquidsolution form was developed. Microalgae can be grown in heterotrophic,mixotrophic, and phototrophic conditions. Culturing microalgae inheterotrophic conditions comprises supplying organic carbon (e.g.,acetic acid, acetate, glucose) to cells in an aqueous culture mediumcomprising trace metals and nutrients (e.g., nitrogen, phosphorus).Culturing microalgae in mixotrophic conditions comprises supplying lightand organic carbon (e.g., acetic acid, acetate, glucose) to cells in anaqueous culture medium comprising trace metals and nutrients (e.g.,nitrogen, phosphorus). Culturing microalgae in phototrophic conditionscomprises supplying light and inorganic carbon (e.g., carbon dioxide) tocells in an aqueous culture medium comprising trace metals and nutrients(e.g., nitrogen, phosphorus).

In some embodiments, the microalgae cells can be harvested from aculture and used as whole cells in a liquid composition for applicationto seeds and plants, while in other embodiments the harvested microalgaecells can be subjected to downstream processing and the resultingbiomass or extract can be used in a dried composition (e.g., powder,pellet) or a liquid composition (e.g., suspension, solution) forapplication to plants, soil, or a combination thereof. Non-limitingexamples of downstream processing comprise: drying the cells, lysing thecells, and subjecting the harvested cells to a solvent or supercriticalcarbon dioxide extraction process to isolate an oil or protein. In someembodiments, the extracted (i.e., residual) biomass remaining from anextraction process can be used alone or in combination with othermicroalgae or extracts in a liquid composition for application toplants, soil, or a combination thereof. By subjecting the microalgae toan extraction process the resulting biomass is transformed from anatural whole state to a lysed condition where the cell is missing asignificant amount of the natural components, thus differentiating theextracted microalgae biomass from that which is found in nature.Excreted products from the microalgae can also be isolated from amicroalgae culture using downstream processing methods.

In some embodiments, microalgae can be the predominant active ingredientsource in the composition. In some embodiments, the microalgaepopulation of the composition can include whole biomass, substantiallyextracted biomass, excreted products (e.g., EPS), extracted protein, orextracted oil. In some embodiments, microalgae include at least 99% ofthe active ingredient sources of the composition. In some embodiments,microalgae include at least 95% of the active ingredient sources of thecomposition. In some embodiments, microalgae include at least 90% of theactive ingredient sources of the composition. In some embodiments,microalgae include at least 80% of the active ingredient sources of thecomposition. In some embodiments, microalgae include at least 70% of theactive ingredient sources of the composition. In some embodiments,microalgae include at least 60% of the active ingredient sources of thecomposition. In some embodiments, microalgae include at least 50% of theactive ingredient sources of the composition. In some embodiments,microalgae include at least 40% of the active ingredient sources of thecomposition. In some embodiments, microalgae include at least 30% of theactive ingredient sources of the composition. In some embodiments,microalgae include at least 20% of the active ingredient sources of thecomposition. In some embodiments, microalgae include at least 10% of theactive ingredient sources of the composition. In some embodiments,microalgae include at least 5% of the active ingredient sources of thecomposition. In some embodiments, microalgae include at least 1% of theactive ingredient sources of the composition. In some embodiments, thecomposition lacks any detectable amount of any other active ingredientsource other than microalgae.

In some embodiments, microalgae biomass, excreted products, or extractscan also be mixed with biomass or extracts from other plants,microalgae, macroalgae, seaweeds, and kelp. In some embodiments,microalgae biomass, excreted products, or extracts can also be mixedwith fish oil. Non-limiting examples of other plants, macroalgae,seaweeds, and kelp fractions that can be combined with microalgae cellscan include species of Lemna, Gracilaria, Kappaphycus, Ascophyllum,Macrocystis, Fucus, Laminaria, Sargassum, Turbinaria, and Durvilea. Infurther embodiments, the extracts can comprise, but are not limited to,liquid extract from a species of Kappaphycus. In some embodiments, theextracts can include 50% or less by volume of the composition. In someembodiments, the extracts can include 40% or less by volume of thecomposition. In some embodiments, the extracts can include 30% or lessby volume of the composition. In some embodiments, the extracts caninclude 20% or less by volume of the composition. In some embodiments,the extracts can include 10% or less by volume of the composition. Insome embodiments, the extracts can include 5% or less by volume of thecomposition. In some embodiments, the extracts can include 4% or less byvolume of the composition. In some embodiments, the extracts can include3% or less by volume of the composition. In some embodiments, theextracts can include 2% or less by volume of the composition. In someembodiments, the extracts can include 1% or less by volume of thecomposition.

The term “microalgae” refers to microscopic single cell organisms suchas microalgae, cyanobacteria, algae, diatoms, dinoflagellates,freshwater organisms, marine organisms, or other similar single cellorganisms capable of growth in phototrophic, mixotrophic, orheterotrophic culture conditions.

In some embodiments, microalgae biomass, excreted product, or extractscan also be sourced from multiple types of microalgae, to make acomposition that is beneficial when applied to plants or soil.Non-limiting examples of microalgae that can be used in the compositionsand methods of the present invention include microalgae in the classes:Eustigmatophyceae, Chlorophyceae, Prasinophyceae, Haptophyceae,Cyanidiophyceae, Prymnesiophyceae, Porphyridiophyceae,Labyrinthulomycetes, Trebouxiophyceae, Bacillariophyceae, andCyanophyceae. The class Cyanidiophyceae includes species of Galdieria.The class Chlorophyceae includes species of Haematococcus, Scenedesmus,Chlamydomonas, and Micractinium. The class Prymnesiophyceae includesspecies of Isochrysis and Pavlova. The class Eustigmatophyceae includesspecies of Nannochloropsis. The class Porphyridiophyceae includesspecies of Porphyridium. The class Labyrinthulomycetes includes speciesof Schizochytrium and Aurantiochytrium. The class Prasinophyceaeincludes species of Tetraselmis. The class Trebouxiophyceae includesspecies of Chlorella and Botryococcus. The class Bacillariophyceaeincludes species of Phaeodactylum. The class Cyanophyceae includesspecies of Spirulina.

Non-limiting examples of microalgae genus and species that can be usedin the compositions and methods of the present invention include:Achnanthes orientalis, Agmenellum spp., Amphiprora hyaline, Amphoracoffeiformis, Amphora coffeiformis var. linea, Amphora coffeiformis var.punctata, Amphora coffeiformis var. taylori, Amphora coffeiformis var.tenuis, Amphora delicatissima, Amphora delicatissima var. capitata,Amphora sp., Anabaena, Ankistrodesmus, Ankistrodesmus falcatus,Aurantiochytrium sp., Boekelovia hooglandii, Borodinella sp.,Botryococcus braunii, Botryococcus sudeticus, Bracteococcus minor,Bracteococcus medionucleatus, Carteria, Chaetoceros gracilis,Chaetoceros muelleri, Chaetoceros muelleri var. subsalsum, Chaetocerossp., Chlamydomonas sp., Chlamydomas perigranulata, Chlorella anitrata,Chlorella antarctica, Chlorella aureoviridis, Chlorella Candida,Chlorella capsulate, Chlorella desiccate, Chlorella ellipsoidea,Chlorella emersonii, Chlorella fusca, Chlorella fusca var. vacuolate,Chlorella glucotropha, Chlorella infusionum, Chlorella infusionum var.actophila, Chlorella infusionum var. auxenophila, Chlorella kessleri,Chlorella lobophora, Chlorella luteoviridis, Chlorella luteoviridis var.aureoviridis, Chlorella luteoviridis var. lutescens, Chlorella miniata,Chlorella minutissima, Chlorella mutabilis, Chlorella nocturna,Chlorella ovalis, Chlorella parva, Chlorella photophila, Chlorellapringsheimii, Chlorella protothecoides, Chlorella protothecoides var.acidicola, Chlorella regularis, Chlorella regularis var. minima,Chlorella regularis var. umbricata, Chlorella reisiglii, Chlorellasaccharophila, Chlorella saccharophila var. ellipsoidea, Chlorellasauna, Chlorella simplex, Chlorella sorokiniana, Chlorella sp.,Chlorella sphaerica, Chlorella stigmatophora, Chlorella vanniellii,Chlorella vulgaris, Chlorella vulgaris fo. tertia, Chlorella vulgarisvar. autotrophica, Chlorella vulgaris var. viridis, Chlorella vulgarisvar. vulgaris, Chlorella vulgaris var. vulgaris fo. tertia, Chlorellavulgaris var. vulgaris fo. viridis, Chlorella xanthella, Chlorellazofingiensis, Chlorella trebouxioides, Chlorella vulgaris, Chlorococcuminfusionum, Chlorococcum sp., Chlorogonium, Chroomonas sp.,Chrysosphaera sp., Cricosphaera sp., Crypthecodinium cohnii, Cryptomonassp., Cyclotella cryptica, Cyclotella meneghiniana, Cyclotella sp.,Dunaliella sp., Dunaliella bardawil, Dunaliella bioculata, Dunaliellagranulate, Dunaliella maritime, Dunaliella minuta, Dunaliella parva,Dunaliella peircei, Dunaliella primolecta, Dunaliella salina, Dunaliellaterricola, Dunaliella tertiolecta, Dunaliella viridis, Dunaliellatertiolecta, Eremosphaera viridis, Eremosphaera sp., Ellipsoidon sp.,Euglena spp., Franceia sp., Fragilaria crotonensis, Fragilaria sp.,Galdieria sp., Gleocapsa sp., Gloeothamnion sp., Haematococcuspluvialis, Hymenomonas sp., Isochrysis a.ff galbana, Isochrysis galbana,Lepocinclis, Micractinium, Monoraphidium minutum, Monoraphidium sp.,Nannochloris sp., Nannochloropsis salina, Nannochloropsis sp., Naviculaacceptata, Navicula biskanterae, Navicula pseudotenelloides, Naviculapelliculosa, Navicula saprophila, Navicula sp., Nephrochloris sp.,Nephroselmis sp., Nitschia communis, Nitzschia alexandrina, Nitzschiaclosterium, Nitzschia communis, Nitzschia dissipata, Nitzschiafrustulum, Nitzschia hantzschiana, Nitzschia inconspicua, Nitzschiaintermedia, Nitzschia microcephala, Nitzschia pusilla, Nitzschia pusillaelliptica, Nitzschia pusilla monoensis, Nitzschia quadrangular,Nitzschia sp., Ochromonas sp., Oocystis parva, Oocystis pusilla,Oocystis sp., Oscillatoria limnetica, Oscillatoria sp., Oscillatoriasubbrevis, Parachlorella kessleri, Pascheria acidophila, Pavlova sp.,Phaeodactylum tricomutum, Phagus, Phormidium, Platymonas sp.,Pleurochrysis camerae, Pleurochrysis dentate, Pleurochrysis sp.,Porphyridium sp., Prototheca wickerhamii, Prototheca stagnora,Prototheca portoricensis, Prototheca moriformis, Prototheca zopfii,Pseudochlorella aquatica, Pyramimonas sp., Pyrobotrys, Rhodococcusopacus, Sarcinoid chrysophyte, Scenedesmus armatus, Schizochytrium,Spirogyra, Spirulina platensis, Stichococcus sp., Synechococcus sp.,Synechocystisf, Tagetes erecta, Tagetes patula, Tetraedron, Tetraselmissp., Tetraselmis suecica, Thalassiosira weissflogii, and Viridiellafridericiana.

Analysis of the DNA sequence of the strain of Chlorella sp. described inthe specification was done in the NCBI 18s rDNA reference database atthe Culture Collection of Algae at the University of Cologne (CCAC)showed substantial similarity (i.e., greater than 95%) with multipleknown strains of Chlorella and Micractinium. Those of skill in the artwill recognize that Chlorella and Micractinium appear closely related inmany taxonomic classification trees for microalgae, and strains andspecies may be re-classified from time to time. Thus, for referencesthroughout the instant specification for Chlorella sp., it is recognizedthat microalgae strains in related taxonomic classifications withsimilar characteristics to the reference Chlorella sp. strain wouldreasonably be expected to produce similar results.

Additionally, taxonomic classification has also been in flux fororganisms in the genus Schizochytrium. Some organisms previouslyclassified as Schizochytrium have been reclassified as Aurantiochytrium,Thraustochytrium, or Oblongichytrium. See Yokoyama et al. Taxonomicrearrangement of the genus Schizochytrium [sensu lato] based onmorphology, chemotaxonomic characteristics, and 18S rRNA gene phylogeny(Thrausochytriaceae, Labyrinthulomycetes): emendation for Schizochytriumand erection of Aurantiochytrium and Oblongichytrium gen. nov.Mycoscience (2007) 48:199-211. Those of skill in the art will recognizethat Schizochytrium, Aurantiochytrium, Thraustochytrium, andOblongichytrium appear closely related in many taxonomic classificationtrees for microalgae, and strains and species may be re-classified fromtime to time. Thus, for references throughout the instant specificationfor Schizochytrium, it is recognized that microalgae strains in relatedtaxonomic classifications with similar characteristics to Schizochytriumwould reasonably be expected to produce similar results.

By artificially controlling aspects of the microalgae culturing processsuch as the organic carbon feed (e.g., acetic acid, acetate), oxygenlevels, pH, and light, the culturing process differs from the culturingprocess that microalgae experiences in nature. In addition tocontrolling various aspects of the culturing process, intervention byhuman operators or automated systems occurs during the non-axenicmixotrophic culturing of microalgae through contamination controlmethods to prevent the microalgae from being overrun and outcompeted bycontaminating organisms (e.g., fungi, bacteria). Contamination controlmethods for microalgae cultures are known in the art and such suitablecontamination control methods for non-axenic mixotrophic microalgaecultures are disclosed in WO2014/074769A2 (Ganuza, et al.), herebyincorporated by reference. By intervening in the microalgae culturingprocess, the impact of the contaminating microorganisms can be mitigatedby suppressing the proliferation of containing organism populations andthe effect on the microalgal cells (e.g., lysing, infection, death,clumping). Thus, through artificial control of aspects of the culturingprocess and intervening in the culturing process with contaminationcontrol methods, the microalgae culture produced as a whole and used inthe described inventive compositions differs from the culture thatresults from a microalgae culturing process that occurs in nature.

During the mixotrophic culturing process the microalgae culture can alsoinclude cell debris and compounds excreted from the microalgae cellsinto the culture medium. The output of the microalgae mixotrophicculturing process provides the active ingredient for composition that isapplied to plants for improving yield and quality without separateaddition to or supplementation of the composition with other activeingredients not found in the mixotrophic microalgae whole cells andaccompanying culture medium from the mixotrophic culturing process suchas, but not limited to: microalgae extracts, macroalgae, macroalgaeextracts, liquid fertilizers, granular fertilizers, mineral complexes(e.g., calcium, sodium, zinc, manganese, cobalt, silicon), fungi,bacteria, nematodes, protozoa, digestate solids, chemicals (e.g.,ethanolamine, borax, boric acid), humic acid, nitrogen and nitrogenderivatives, phosphorus rock, pesticides, herbicides, insecticides,enzymes, plant fiber (e.g., coconut fiber).

In some embodiments, the microalgae can be previously frozen and thawedbefore inclusion in the liquid composition. In some embodiments, themicroalgae may not have been subjected to a previous freezing or thawingprocess. In some embodiments, the microalgae whole cells have not beensubjected to a drying process. The cell walls of the microalgae of thecomposition have not been lysed or disrupted, and the microalgae cellshave not been subjected to an extraction process or process thatpulverizes the cells. The microalgae whole cells are not subjected to apurification process for isolating the microalgae whole cells from theaccompanying constituents of the culturing process (e.g., tracenutrients, residual organic carbon, bacteria, cell debris, cellexcretions), and thus the whole output from the microalgae culturingprocess comprising whole microalgae cells, culture medium, cellexcretions, cell debris, bacteria, residual organic carbon, and tracenutrients, is used in the liquid composition for application to plants.In some embodiments, the microalgae whole cells and the accompanyingconstituents of the culturing process are concentrated in thecomposition. In some embodiments, the microalgae whole cells and theaccompanying constituents of the culturing process are diluted in thecomposition to a low concentration. The microalgae whole cells of thecomposition are not fossilized. In some embodiments, the microalgaewhole cells are not maintained in a viable state in the composition forcontinued growth after the method of using the composition in a soil orfoliar application. In some embodiments, the microalgae base compositioncan be biologically inactive after the composition is prepared. In someembodiments, the microalgae base composition can be substantiallybiologically inactive after the composition is prepared. In someembodiments, the microalgae base composition can increase in biologicalactivity after the prepared composition is exposed to air.

In some embodiments, a liquid composition can include low concentrationsof bacteria contributing to the solids percentage of the composition inaddition to the microalgae cells. Examples of bacteria found innon-axenic mixotrophic conditions can be found in WO2014/074769A2(Ganuza, et al.), hereby incorporated by reference. A live bacteriacount can be determined using methods known in the art such as platecounts, plates counts using Petrifilm available from 3M (St. Paul,Minn.), spectrophotometric (turbidimetric) measurements, visualcomparison of turbidity with a known standard, direct cell counts undera microscope, cell mass determination, and measurement of cellularactivity. Live bacteria counts in a non-axenic mixotrophic microalgaeculture can range from I0⁴ to I0⁹ CFU/mL, and can depend oncontamination control measures taken during the culturing of themicroalgae. The level of bacteria in the composition can be determinedby an aerobic plate count which quantifies aerobic colony forming units(CFU) in a designated volume. In some embodiments, the compositionincludes an aerobic plate count of 40,000-400,000 CFU/mL. In someembodiments, the composition includes an aerobic plate count of40,000-100,000 CFU/mL. In some embodiments, the composition includes anaerobic plate count of 100,000-200,000 CFU/mL. In some embodiments, thecomposition includes an aerobic plate count of 200,000-300,000 CFU/mL.In some embodiments, the composition includes an aerobic plate count of300,000-400,000 CFU/mL.

In some embodiments, the microalgae based composition can besupplemented with a supplemental nutrient such as nitrogen, phosphorus,or potassium to increase the levels within the composition to at least1% of the total composition (i.e., addition of N, P, or K to increaselevels at least 1-0-0, 0-1-0, 0-0-1, or combinations thereof). In someembodiments, the microalgae composition can be supplemented withnutrients such as, but not limited to, calcium, magnesium, silicon,sulfur, iron, manganese, zinc, copper, boron, molybdenum, chlorine,sodium, aluminum, vanadium, nickel, cerium, dysprosium, erbium,europium, gadolinium, holmium, lanthanum, lutetium, neodymium,praseodymium, promethium, samarium, scandium, terbium, thulium,ytterbium, and yttrium. In some embodiments, the supplemented nutrientis not uptaken, chelated, or absorbed by the microalgae. In someembodiments, the concentration of the supplemental nutrient can include1-50 g per 100 g of the composition.

A liquid composition comprising microalgae can be stabilized by heatingand cooling in a pasteurization process. As shown in the Examples, theinventors found that the active ingredients of the microalgae basedcomposition maintained effectiveness in at least one characteristic of aplant after being subjected to the heating and cooling of apasteurization process. In other embodiments, liquid compositions withwhole cells or processed cells (e.g., dried, lysed, extracted) ofmicroalgae cells may not need to be stabilized by pasteurization. Forexample, microalgae cells that have been processed, such as by drying,lysing, and extraction, or extracts can include such low levels ofbacteria that a liquid composition can remain stable without beingsubjected to the heating and cooling of a pasteurization process.

In some embodiments, the composition can be heated to a temperature inthe range of 50-130° C. In some embodiments, the composition can beheated to a temperature in the range of 55-65° C. In some embodiments,the composition can be heated to a temperature in the range of 58-62° C.In some embodiments, the composition can be heated to a temperature inthe range of 50-60° C. In some embodiments, the composition can beheated to a temperature in the range of 60-90° C. In some embodiments,the composition can be heated to a temperature in the range of 70-80° C.In some embodiments, the composition can be heated to a temperature inthe range of 100-50° C. In some embodiments, the composition can beheated to a temperature in the range of 120-130° C.

In some embodiments, the composition can be heated for a time period inthe range of 1-150 minutes. In some embodiments, the composition can beheated for a time period in the range of 110-130 minutes. In someembodiments, the composition can be heated for a time period in therange of 90-100 minutes. In some embodiments, the composition can beheated for a time period in the range of 100-110 minutes. In someembodiments, the composition can be heated for a time period in therange of 110-120 minutes. In some embodiments, the composition can beheated for a time period in the range of 120-130 minutes. In someembodiments, the composition can be heated for a time period in therange of 130-140 minutes. In some embodiments, the composition can beheated for a time period in the range of 140-150 minutes. In someembodiments, the composition is heated for less than 15 min. In someembodiments, the composition is heated for less than 2 min.

After the step of heating or subjecting the liquid composition to hightemperatures is complete, the compositions can be cooled at any rate toa temperature that is safe to work with. In one non-limiting embodiment,the composition can be cooled to a temperature in the range of 35-45° C.In some embodiments, the composition can be cooled to a temperature inthe range of 36-44° C. In some embodiments, the composition can becooled to a temperature in the range of 37-43° C. In some embodiments,the composition can be cooled to a temperature in the range of 38-42° C.In some embodiments, the composition can be cooled to a temperature inthe range of 39-41° C. In further embodiments, the pasteurizationprocess can be part of a continuous production process that alsoinvolves packaging, and thus the liquid composition can be packaged(e.g., bottled) directly after the heating or high temperature stagewithout a cooling step.

In some embodiments, the composition can include 5-30% solids by weightof microalgae cells (i.e., 5-30 g of microalgae cells/100 mL of theliquid composition). In some embodiments, the composition can include5-20% solids by weight of microalgae cells. In some embodiments, thecomposition can include 5-15% solids by weight of microalgae cells. Insome embodiments, the composition can include 5-10% solids by weight ofmicroalgae cells. In some embodiments, the composition can include10-20% solids by weight of microalgae cells. In some embodiments, thecomposition can include 10-20% solids by weight of microalgae cells. Insome embodiments, the composition can include 20-30% solids by weight ofmicroalgae cells. In some embodiments, further dilution of themicroalgae cells percent solids by weight can occur before applicationfor low concentration applications of the composition.

In some embodiments, the composition can include less than 1% by weightof microalgae biomass or extracts (i.e., less than 1 g of microalgaederived product/100 mL of the liquid composition). In some embodiments,the composition can include less than 0.9% by weight of microalgaebiomass or extracts. In some embodiments, the composition can includeless than 0.8% by weight of microalgae biomass or extracts. In someembodiments, the composition can include less than 0.7% by weight ofmicroalgae biomass or extracts. In some embodiments, the composition caninclude less than 0.6% by weight of microalgae biomass or extracts. Insome embodiments, the composition can include less than 0.5% by weightof microalgae biomass or extracts. In some embodiments, the compositioncan include less than 0.4% by weight of microalgae biomass or extracts.In some embodiments, the composition can include less than 0.3% byweight of microalgae biomass or extracts. In some embodiments, thecomposition can include less than 0.2% by weight of microalgae biomassor extracts. In some embodiments, the composition can include less than0.1% by weight of microalgae biomass or extracts. In some embodiments,the composition can include at least 0.0001% by weight of microalgaebiomass or extracts. In some embodiments, the composition can include atleast 0.001% by weight of microalgae biomass or extracts. In someembodiments, the composition can include at least 0.01% by weight ofmicroalgae biomass or extracts. In some embodiments, the composition caninclude at least 0.1% by weight of microalgae biomass or extracts. Insome embodiments, the composition can include 0.0001-1% by weight ofmicroalgae biomass or extracts. In some embodiments, the composition caninclude 0.0001-0.001% by weight of microalgae biomass or extracts. Insome embodiments, the composition can include 0.001-0.01% by weight ofmicroalgae biomass or extracts. In some embodiments, the composition caninclude 0.01-0.1% by weight of microalgae biomass or extracts. In someembodiments, the composition can include 0.1-1% by weight of microalgaebiomass or extracts.

In some embodiments, an application concentration of 0.1% of microalgaebiomass or extract equates to 0.04 g of microalgae biomass or extract in40 mL of a composition. While the desired application concentration to aplant can be 0.1% of microalgae biomass or extract, the composition canbe packaged as a 10% concentration (0.4 mL in 40 mL of a composition).Thus, a desired application concentration of 0.1% would require 6,000 mLof the 10% microalgae biomass or extract in the 100 gallons of waterapplied to the assumption of 15,000 plants in an acre, which isequivalent to an application rate of about 1.585 gallons per acre. Insome embodiments, a desired application concentration of 0.01% ofmicroalgae biomass or extract using a 10% concentration compositionequates to an application rate of about 0.159 gallons per acre. In someembodiments, a desired application concentration of 0.001% of microalgaebiomass or extract using a 10% concentration composition equates to anapplication rate of about 0.016 gallons per acre. In some embodiments, adesired application concentration of 0.0001% of microalgae biomass orextract using a 10% concentration composition equates to an applicationrate of about 0.002 gallons per acre.

In another non-limiting embodiment, correlating the application of themicroalgae biomass or extract on a per plant basis using the assumptionof 15,000 plants per acre, the composition application rate of 1 gallonper acre is equal to about 0.25 mL per plant=0.025 g per plant=25 mg ofmicroalgae biomass or extract per plant. The water requirementassumption of 100 gallons per acre is equal to about 35 mL of water perplant. Therefore, 0.025 g of microalgae biomass or extract in 35 mL ofwater is equal to about 0.071 g of microalgae biomass or extract per 100mL of composition equates to about a 0.07% application concentration. Insome embodiments, the microalgae biomass or extract based compositioncan be applied at a rate in a range as low as about 0.001-10 gallons peracre, or as high as up to 150 gallons per acre.

In some of the embodiments and Examples below, the applications wereperformed using a 10% solids solution by weight microalgae composition.For these field trials, the rates are indicated in gal/acre and theamount of carrier water would be determined according to userpreference. For field trials, the application rate may range between0.25 gal/acre-2 gal/acre. In the field trials, where the applicationrate of the microalgae composition is 0.25 gal/acre, the equivalentexpressed in total grams of solid microalgae would be 100 gmicroalgae/acre; wherein the application rate of the microalgaecomposition is 0.4 gal/acre, the equivalent expressed in total grams ofsolid microalgae would be 160 g microalgae/acre; where the applicationrate of the microalgae composition is 0.5 gal/acre, the equivalentexpressed in total grams of solid microalgae would be 200 gmicroalgae/acre; where the application rate of the microalgaecomposition is 1.0 gal/acre, the equivalent expressed in total grams ofsolid microalgae would be 400 g microalgae/acre; and where theapplication rate of the microalgae composition is 2.0 gal/acre, theequivalent expressed in total grams of solid microalgae would be 800 gmicroalgae/acre.

Overall, as shown in the embodiments and Examples below, the microalgaecomposition may comprise between 100 g-800 g per acre, as it is commonpractice for growers to use between 100-250 gallons of liquid carriervolume/acre. It should be clearly understood, however, thatmodifications to the amount of microalgae per acre may be adjustedupwardly or downwardly to compensate for greater than 250 gallons ofliquid carrier volume/acre or less than 100 gallons of liquid carriervolume/acre.

In some embodiments, stabilizing means that are not active regarding theimprovement of plant germination, emergence, maturation, quality, andyield, but instead aid in stabilizing the composition can be added toprevent the proliferation of unwanted microorganisms (e.g., yeast, mold)and prolong shelf life. Such inactive but stabilizing means can includean acid, such as but not limited to phosphoric acid or citric acid, anda yeast and mold inhibitor, such as but not limited to potassiumsorbate. In some embodiments, the stabilizing means are suitable forplants and do not inhibit the growth or health of the plant. In thealternative, the stabilizing means can contribute to nutritionalproperties of the liquid composition, such as but not limited to, thelevels of nitrogen, phosphorus, or potassium.

In some embodiments, the composition can include between 0.5-1.5%phosphoric acid. In other embodiments, the composition may comprise lessthan 0.5% phosphoric acid. In some embodiments, the composition caninclude 0.01-0.3% phosphoric acid. In some embodiments, the compositioncan include 0.05-0.25% phosphoric acid. In some embodiments, thecomposition can include 0.01-0.1% phosphoric acid. In some embodiments,the composition can include 0.1-0.2% phosphoric acid. In someembodiments, the composition can include 0.2-0.3% phosphoric acid. Insome embodiments, the composition can include less than 0.3% citricacid.

In some embodiments, the composition can include 1.0-2.0% citric acid.In other embodiments, the composition can include 0.01-0.3% citric acid.In some embodiments, the composition can include 0.05-0.25% citric acid.In some embodiments, the composition can include 0.01-0.1% citric acid.In some embodiments, the composition can include 0.1-0.2% citric acid.In some embodiments, the composition can include 0.2-0.3% citric acid.

In some embodiments, the composition can include less than 0.5%potassium sorbate. In some embodiments, the composition can include0.01-0.5% potassium sorbate. In some embodiments, the composition caninclude 0.05-0.4% potassium sorbate. In some embodiments, thecomposition can include 0.01-0.1% potassium sorbate. In someembodiments, the composition can include 0.1-0.2% potassium sorbate. Insome embodiments, the composition can include 0.2-0.3% potassiumsorbate. In some embodiments, the composition can include 0.3-0.4%potassium sorbate. In some embodiments, the composition can include0.4-0.5% potassium sorbate.

The present invention involves the use of a microalgae composition.Microalgae compositions, methods of preparing liquid microalgaecompositions, and methods of applying the microalgae compositions toplants are disclosed in WO2017/218896A1 (Shinde et al.) entitledMicroalgae-Based Composition, and Methods of its Preparation andApplication to Plants, which is incorporated herein in full byreference. In one or more embodiments, the microalgae composition maycomprise approximately 10%40.5% w/w of Chlorella microalgae cells. Inone or more embodiments, the microalgae composition may also compriseone of more stabilizers, such as potassium sorbate, phosphoric acid,ascorbic acid, sodium benzoate, citric acid, or the like, or anycombination thereof. For example, in one or more embodiments, themicroalgae composition may comprise approximately 0.3% w/w of potassiumsorbate or another similar compound to stabilize its pH and may furthercomprise approximately 0.5-1.5% w/w phosphoric acid or another similarcompound to prevent the growth of contaminants. As a further example, inone or more embodiments where it is desired to use an OMRI (OrganicMaterials Review Institute) certified organic composition, themicroalgae composition may comprise 1.0-2.0% w/w citric acid tostabilize its pH, and may not contain potassium sorbate or phosphoricacid. In one or more embodiments, the pH of the microalgae compositionmay be stabilized to between 3.0-4.0.

In some embodiments and Examples below, the microalgae composition maybe referred to as PHYCOTERRA®. The PHYCOTERRA® Chlorella microalgaecomposition is a microalgae composition comprising Chlorella. ThePHYCOTERRA® Chlorella microalgae composition treatments were prepared bygrowing the Chlorella in non-axenic acetic acid supplied mixotrophicconditions, increasing the concentration of Chlorella using acentrifuge, pasteurizing the concentrated Chlorella at between 65°C.-75° C. for between 90-150 minutes, adding potassium sorbate andphosphoric acid to stabilize the pH of the Chlorella, and then adjustingthe whole biomass treatment to the desired concentration. ThePHYCOTERRA® Chlorella microalgae composition may comprise approximately10% w/w of Chlorella microalgae cells. Furthermore, the PHYCOTERRA®Chlorella microalgae composition may comprise between approximately 0.3%potassium sorbate and between approximately 0.5%-1.5% phosphoric acid tostabilize the pH of the Chlorella to between 3.0-4.0 and 88.2%-89.2%water. It should be clearly understood, however, that other variationsof the PHYCOTERRA® Chlorella microalgae composition, includingvariations in the microalgae strains, variations in the stabilizers,and/or variations in the % composition of each component may be used andmay achieve similar results.

In some embodiments and Examples below, the microalgae composition maybe an OMRI certified microalgae composition referred to as TERRENE®. TheOMRI certified TERRENE® Chlorella microalgae composition is a microalgaecomposition comprising Chlorella. The OMRI certified TERRENE® Chlorellamicroalgae composition treatments were prepared by growing the Chlorellain non-axenic acetic acid supplied mixotrophic conditions, increasingthe concentration of Chlorella using a centrifuge, pasteurizing theconcentrated Chlorella at between 65° C.-75° C. for between 90-150minutes, adding citric acid to stabilize the pH of the Chlorella, andthen adjusting the whole biomass treatment to the desired concentration.The OMRI certified TERRENE® Chlorella microalgae composition maycomprise approximately 10% w/w of Chlorella microalgae cells.Furthermore, the OMRI certified TERRENE® Chlorella microalgaecomposition may comprise between approximately 0.5%-2.0% citric acid tostabilize the pH of the Chlorella to between 3.0-4.0 and 88%-89.5%water. It should be clearly understood, however, that other variationsof the OMRI certified TERRENE® Chlorella microalgae composition,including variations in the microalgae strains, variations in thestabilizers, and/or variations in the % composition of each componentmay be used and may achieve similar results.

In some embodiments and Examples below, the microalgae composition maybe an OMRI certified microalgae composition referred to as OMRIcertified TERRENE® Chlorella pasteurized at 65° C. microalgaecomposition or as TERRENE65. The OMRI certified TERRENE® Chlorellapasteurized at 65° C. microalgae composition is a microalgae compositioncomprising Chlorella. The OMRI certified TERRENE® Chlorella pasteurizedat 65° C. microalgae composition treatments were prepared by growing theChlorella in non-axenic acetic acid supplied mixotrophic conditions,increasing the concentration of Chlorella using a centrifuge,pasteurizing the concentrated Chlorella at 65° C. for between 90-150minutes, adding citric acid to stabilize the pH of the Chlorella, andthen adjusting the whole biomass treatment to the desired concentration.The OMRI certified TERRENE® Chlorella pasteurized at 65° C. microalgaecomposition may comprise approximately 10% w/w of Chlorella microalgaecells. Furthermore, the OMRI certified TERRENE® Chlorella pasteurized at65° C. microalgae composition may comprise between approximately0.5%-2.0% citric acid to stabilize the pH of the Chlorella to between3.0-4.0 and 88-89.5% water. It should be clearly understood, however,that other variations of the OMRI certified TERRENE® Chlorellapasteurized at 65° C. microalgae composition, including variations inthe microalgae strains, variations in the stabilizers, variations in thepasteurization temperature, and/or variations in the % composition ofeach component may be used and may achieve similar results.

In some embodiments and Examples below, the microalgae composition maybe an OMRI certified microalgae composition referred to as OMRIcertified TERRENE® Chlorella pasteurized at 90° C. microalgaecomposition or as TERRENE90. The OMRI certified TERRENE® Chlorellapasteurized at 90° C. microalgae composition is a microalgae compositioncomprising Chlorella. The OMRI certified TERRENE® Chlorella pasteurizedat 90° C. microalgae composition treatments were prepared by growing theChlorella in non-axenic acetic acid supplied mixotrophic conditions,increasing the concentration of Chlorella using a centrifuge,pasteurizing the concentrated Chlorella at 90° C. for between 90-150minutes, adding citric acid to stabilize the pH of the Chlorella, andthen adjusting the whole biomass treatment to the desired concentration.The OMRI certified TERRENE® Chlorella pasteurized at 90° C. microalgaecomposition may comprise approximately 10% w/w of Chlorella microalgaecells. Furthermore, the OMRI certified TERRENE® Chlorella pasteurized at90° C. microalgae composition may comprise between approximately0.5%-2.0% citric acid to stabilize the pH of the Chlorella to between3.0-4.0 and 88-89.5% water. It should be clearly understood that othervariations of the OMRI certified TERRENE® Chlorella pasteurized at 90°C. microalgae composition, including variations in the microalgaestrains, variations in the stabilizers, variations in the pasteurizationtemperature, and/or variations in the % composition of each componentmay be used and may achieve similar results.

In some embodiments and Examples below, the microalgae composition maybe referred to as Aurantiochytrium acetophilum HS399 whole biomass (WB)or HS399 WB. The Aurantiochytrium acetophilum HS399 whole biomass (WB)microalgae composition is a microalgae composition comprisingAurantiochytrium acetophilum HS399. The Aurantiochytrium acetophilumHS399 whole biomass (WB) microalgae composition treatments were preparedby growing the Aurantiochytrium acetophilum HS399 microalgae innon-axenic acetic acid supplied heterotrophic conditions, increasing theconcentration of Aurantiochytrium acetophilum HS399 using a centrifuge,pasteurizing the concentrated Aurantiochytrium acetophilum HS399 atbetween 65° C.-75° C. for between 90-150 minutes, adding approximately0.3% w/w of potassium sorbate and between approximately 0.5-1.5%phosphoric acid to stabilize the pH of the Aurantiochytrium acetophilumHS399 to between 3.0-4.0, and then adjusting the whole biomass to adesired concentration. It should be clearly understood that othervariations of the Aurantiochytrium acetophilum HS399 whole biomass (WB)microalgae composition, including variations in the microalgae strains,variations in the stabilizers, variations in the pasteurizationtemperature, and/or variations in the % composition of each componentmay be used and may achieve similar results.

In some embodiments and Examples below, the microalgae composition maybe referred to as Aurantiochytrium acetophilum HS399 washed wholebiomass (WB washed). The Aurantiochytrium acetophilum HS399 washed wholebiomass (WB washed) microalgae composition is a microalgae compositioncomprising Aurantiochytrium acetophilum HS399. The Aurantiochytriumacetophilum HS399 washed whole biomass (WB washed) microalgaecomposition treatments were prepared by growing the Aurantiochytriumacetophilum HS399 microalgae in non-axenic acetic acid suppliedheterotrophic conditions, increasing the concentration ofAurantiochytrium acetophilum HS399 using a centrifuge, pasteurizing theconcentrated Aurantiochytrium acetophilum HS399 at between 65° C.-75° C.for between 90-150 minutes, adding approximately 0.3% w/w of potassiumsorbate and between approximately 0.5%-1.5% phosphoric acid to stabilizethe pH of the Aurantiochytrium acetophilum HS399 to between 3.0-4.0, andthen adjusting the whole biomass to a desired concentration. Once theAurantiochytrium acetophilum HS399 microalgae cells were concentratedfrom the harvest, they were washed; i.e. diluted with water in a ratioof 5:1 and centrifuged again in order to remove dissolved material andsmall particles. It should be clearly understood that other variationsof the Aurantiochytrium acetophilum HS399 washed whole biomass (WBwashed) microalgae composition, including variations in the microalgaestrains, variations in the stabilizers, variations in the pasteurizationtemperature, variations in the washing method, and/or variations in the% composition of each component may be used and may achieve similarresults.

In some embodiments and Examples below, the microalgae composition maybe referred to as Aurantiochytrium acetophilum HS399 extracted biomass(EB) or HS399 EB. The Aurantiochytrium acetophilum HS399 extractedbiomass (EB) microalgae composition is a microalgae compositioncomprising Aurantiochytrium acetophilum HS399. The Aurantiochytriumacetophilum HS399 extracted biomass (EB) treatments were prepared bygrowing the Aurantiochytrium acetophilum HS399 microalgae in non-axenicacetic acid supplied heterotrophic conditions, increasing theconcentration of Aurantiochytrium acetophilum HS399 using a centrifuge,pasteurizing the concentrated Aurantiochytrium acetophilum HS399 atbetween 65° C.-75° C. for between 90-150 minutes, adding approximately0.3% w/w of potassium sorbate and between approximately 0.5%-1.5%phosphoric acid to stabilize the pH of the Aurantiochytrium acetophilumHS399 to between 3.0-4.0, processing the Aurantiochytrium acetophilumHS399 with an oat filler in an expeller process to lyse the cells andseparate oil from the residual biomass, and then adjusting the residualbiomass to a desired concentration. It should be clearly understood thatother variations of the Aurantiochytrium acetophilum HS399 extractedbiomass (EB) microalgae composition, including variations in themicroalgae strains, variations in the stabilizers, variations in thepasteurization temperature, variations in the extraction method, and/orvariations in the % composition of each component may be used and mayachieve similar results.

In some embodiments and Examples below, the microalgae composition maybe referred to as a combination 25% Chlorella: 75% HS399 whole biomass(WB) microalgae composition or 25% Chlorella: 75% HS399 WB. Thecombination 25% Chlorella: 75% HS399 whole biomass (WB) microalgaecomposition is a microalgae composition comprising Chlorella andAurantiochytrium acetophilum HS399. For the combination 25% Chlorella:75% HS399 whole biomass (WB) microalgae composition, the Chlorellamicroalgae cells were cultured in outdoor pond reactors in non-axenicacetic acid supplied mixotrophic conditions and the concentration ofChlorella was increased using a centrifuge. The Aurantiochytriumacetophilum HS399 cells were cultured in non-axenic acetic-acid suppliedheterotrophic conditions and the concentration of HS399 was increasedusing a centrifuge. The concentrated Chlorella cells were then combinedwith the concentrated HS399 whole biomass cells and adjusted to thedesired concentration of 25% Chlorella: 75% HS399 whole biomass (WB).The combination 25% Chlorella: 75% HS399 whole biomass (WB) microalgaecomposition was then pasteurized at between 65° C.-75° C. for between90-150 minutes and then stabilized by adding approximately 0.3% w/w ofpotassium sorbate and between approximately 0.5%-1.5% phosphoric acid tostabilize the pH of the 25% Chlorella: 75% HS399 whole biomass (WB)microalgae composition to between 3.0-4.0. It should be clearlyunderstood, however, that other variations of the combination 25%Chlorella: 75% HS399 whole biomass (WB) microalgae composition,including variations in the microalgae strains, variations in thestabilizers, variations in the order of the processing steps (blending,pasteurizing, stabilizing), and/or variations in the % composition ofeach component may be used and may achieve similar results.

In some embodiments and Examples below, the microalgae composition maybe referred to as GP2C. The GP2C Chlorella microalgae compositioncomprised Chlorella. The GP2C Chlorella microalgae compositiontreatments were prepared by growing the Chlorella in non-axenic aceticacid supplied mixotrophic conditions, increasing the concentration ofChlorella using a centrifuge, pasteurizing the concentrated Chlorella atbetween 65° C.-75° C. for between 90-150 minutes, adding potassiumsorbate and phosphoric acid to stabilize the pH of the Chlorella, andthen adjusting the whole biomass treatment to the desired concentration.The GP2C Chlorella microalgae composition may comprise approximately 10%w/w of Chlorella microalgae cells. Furthermore, the GP2C microalgaecomposition may comprise between approximately 0.3% potassium sorbateand between approximately 05%-1.5% phosphoric acid to stabilize the pHof the Chlorella to between 3.0-4.0 and 88.2%-89% water. It should beclearly understood, however, that other variations of the GP2C Chlorellamicroalgae composition, including variations in the microalgae strains,variations in the stabilizers, and/or variations in the % composition ofeach component may be used and may achieve similar results.

In some embodiments and Examples below, the microalgae composition maybe referred to as a combination 25% Chlorella: 75% HS399 extractedbiomass (EB) microalgae composition, a 50% Chlorella: 50% HS399extracted biomass (EB) microalgae composition, a 75% Chlorella: 25%HS399 extracted biomass (EB) microalgae composition, or a combinationGP2C:399 microalgae composition. The combination GP2C:399 microalgaecomposition comprises Chlorella and Aurantiochytrium acetophilum HS399extracted biomass (EB). For the combination GP2C:399 microalgaecomposition, the Chlorella microalgae cells were cultured in outdoorpond reactors in non-axenic acetic acid supplied mixotrophic conditionsand the concentration of Chlorella was increased using a centrifuge; theAurantiochytrium acetophilum HS399 microalgae cells were cultured innon-axenic acetic acid supplied heterotrophic conditions, theconcentration of HS399 was increased using a centrifuge, and the HS399cells were then processed with an oat filler in an expeller process tolyse the cells and separate oil from the residual biomass. Theconcentrated GP2C Chlorella whole biomass microalgae cells and theAurantiochytrium acetophilum HS399 extracted biomass (EB) microalgaecells were blended together to the ratios of 50:50, 25:75, and 75:25,then pasteurized at between 65° C.-75° C. for between 90-150 minutes andthen stabilized by adding approximately 0.3% w/w of potassium sorbateand between approximately 0.5%-1.5% phosphoric acid to stabilize the pHof the 25% Chlorella: 75% HS399 extracted biomass (EB) microalgaecomposition to between 3.0-4.0. It should be clearly understood,however, that other variations of the combination GP2C:399 microalgaecomposition, including variations in the microalgae strains, variationsin the stabilizers, variations in the order of the processing steps(blending, pasteurizing, stabilizing), and/or variations in the %composition of each component may be used and may achieve similarresults.

In some embodiments and Examples below, the microalgae composition maybe referred to as a Greenwater Polyculture (GWP) treatment. GreenwaterPolyculture may be prepared by beginning with a culture of Scenedesmusmicroalgae that is left outdoors in an open pond and harvestedcontinuously over a year. The culture may comprise anywhere from lessthan 50% Scenedesmus to greater than 75% Scenedesmus and theconcentration varies throughout the year. Other algae may colonize inthe GWP as well as other bacteria and microorganisms.

In some embodiments, the composition is a liquid and substantiallyincludes of water. In some embodiments, the composition can include70-99% water. In some embodiments, the composition can include 85-95%water. In some embodiments, the composition can include 70-75% water. Insome embodiments, the composition can include 75-80% water. In someembodiments, the composition can include 80-85% water. In someembodiments, the composition can include 85-90% water. In someembodiments, the composition can include 90-95% water. In someembodiments, the composition can include 95-99% water. The liquid natureand high-water content of the composition facilitates administration ofthe composition in a variety of manners, such as but not limit to:flowing through an irrigation system, flowing through an above grounddrip irrigation system, flowing through a buried drip irrigation system,flowing through a central pivot irrigation system, sprayers, sprinklers,and water cans.

In some embodiments, the liquid composition can be used immediatelyafter formulation, or can be stored in containers for later use. In someembodiments, the composition can be stored out of direct sunlight. Insome embodiments, the composition can be refrigerated. In someembodiments, the composition can be stored at 1-10° C. In someembodiments, the composition can be stored at 1-3° C. In someembodiments, the composition can be stored at 3-50° C. In someembodiments, the composition can be stored at 5-8° C. In someembodiments, the composition can be stored at 8-10° C.

In some embodiments, administration of the liquid composition to soil, aseed or plant can be in an amount effective to produce an enhancedcharacteristic in plants compared to a substantially identicalpopulation of untreated seeds or plants. Such enhanced characteristicscan include accelerated seed germination, accelerated seedlingemergence, improved seedling emergence, improved leaf formation,accelerated leaf formation, improved plant maturation, accelerated plantmaturation, increased plant yield, increased plant growth, increasedplant quality, increased plant health, increased fruit yield, increasedfruit sweetness, increased fruit growth, and increased fruit quality.Non-limiting examples of such enhanced characteristics can includeaccelerated achievement of the hypocotyl stage, accelerated protrusionof a stem from the soil, accelerated achievement of the cotyledon stage,accelerated leaf formation, increased marketable plant weight, increasedmarketable plant yield, increased marketable fruit weight, increasedproduction plant weight, increased production fruit weight, increasedutilization (indicator of efficiency in the agricultural process basedon ratio of marketable fruit to unmarketable fruit), increasedchlorophyll content (indicator of plant health), increased plant weight(indicator of plant health), increased root weight (indicator of planthealth), increased shoot weight (indicator of plant health), increasedplant height, increased thatch height, increased resistance to saltstress, increased plant resistance to heat stress (temperature stress),increased plant resistance to heavy metal stress, increased plantresistance to drought, increased plant resistance to disease, improvedcolor, reduced insect damage, reduced blossom end rot, and reduced sunburn. Such enhanced characteristics can occur individually in a plant,or in combinations of multiple enhanced characteristics.

In some embodiments, a liquid composition can be administered before theseed is planted. In some embodiments, a liquid composition can beadministered at the time the seed is planted. In some embodiments, aliquid composition can be applied by dip treatment of the roots. In someembodiments, a liquid composition can be administered to plants thathave emerged from the ground. In some embodiments, a liquid or driedcomposition can be applied to the soil before, during, or after theplanting of a seed. In some embodiments a liquid or dried compositioncan be applied to the soil before or after a plant emerges from thesoil.

In some embodiments, the volume or mass of the microalgae basedcomposition applied to a seed, seedling, or plant may not increase ordecrease during the growth cycle of the plant (i.e., the amount of themicroalgae composition applied to the plant will not change as the plantgrows larger). In some embodiments, the volume or mass of the microalgaebased composition applied to a seed, seedling, or plant can increaseduring the growth cycle of the plant (i.e., applied on a mass or volumeper plant mass basis to provide more of the microalgae composition asthe plant grows larger). In some embodiments, the volume or mass of themicroalgae based composition applied to a seed, seedling, or plant candecrease during the growth cycle of the plant (i.e., applied on a massor volume per plant mass basis to provide more of the microalgaecomposition as the plant grows larger).

In one non-limiting embodiment, the administration of the compositionmay comprise contacting the foliage of the plant with an effectiveamount of the composition. In some embodiments, the liquid compositionmay be sprayed on the foliage by a hand sprayer, a sprayer on anagriculture implement, or a sprinkler. In some embodiments, thecomposition can be applied to the soil.

The rate of application of the composition at the desired concentrationcan be expressed as a volume per area. In some embodiments, the rate ofapplication of the liquid composition in a foliar application cancomprise a rate in the range of 10-50 gallons/acre. In some embodiments,the rate of application of the liquid composition in a foliarapplication can comprise a rate in the rage of 10-15 gallons/acre. Insome embodiments, the rate of application of the liquid composition in afoliar application can comprise a rate in the range of 15-20gallons/acre. In some embodiments, the rate of application of the liquidcomposition in a foliar application can comprise a rate in the range of20-25 gallons/acre. In some embodiments, the rate of application of theliquid composition in a foliar application can comprise a rate in therange of 25-30 gallons/acre. In some embodiments, the rate ofapplication of the liquid composition in a foliar application cancomprise a rate in the range of 30-35 gallons/acre. In some embodiments,the rate of application of the liquid composition in a foliarapplication can comprise a rate in the range of 35-40 gallons/acre. Insome embodiments, the rate of application of the liquid composition in afoliar application can comprise a rate in the range of 40-45gallons/acre. In some embodiments, the rate of application of the liquidcomposition in a foliar application can comprise a rate in the range of45-50 gallons/acre.

In some embodiments, the rate of application of the liquid compositionin a soil or foliar application can comprise a rate in the range of0.01-10 gallons/acre. In some embodiments, the rate can be 0.12-4%. Insome embodiments, the rate of application of the liquid composition in afoliar application may comprise a rate in the range of 0.01-0.1gallons/acre. In some embodiments, the rate of application of the liquidcomposition in a soil or foliar application may comprise a rate in therange of 0.1-1.0 gallons/acre. In some embodiments, the rate ofapplication of the liquid composition in a foliar application maycomprise a rate in the range of 0.25-2 gallons/acre. In someembodiments, the rate of application of the liquid composition in afoliar application may comprise a rate in the range of 1-2 gallons/acre.In some embodiments, the rate of application of the liquid compositionin a foliar application may comprise a rate in the range of 2-3gallons/acre. In some embodiments, the rate of application of the liquidcomposition in a foliar application may comprise a rate in the range of3-4 gallons/acre. In some embodiments, the rate of application of theliquid composition in a foliar application may comprise a rate in therange of 4-5 gallons/acre. In some embodiments, the rate of applicationof the liquid composition in a foliar application may comprise a rate inthe range of 5-10 gallons/acre.

In some embodiments, the v/v ratio of the composition can be between0.001%-50%. The v/v ratio can be between 0.01-25%. The v/v ratio of thecomposition can be between 0.03-10%.

The frequency of the application of the composition can be expressed asthe number of applications per period of time (e.g., two applicationsper month), or by the period of time between applications (e.g., oneapplication every 21 days). In some embodiments, the plant can becontacted by the composition in a foliar application every 3-28 days. Insome embodiments, the plant can be contacted by the composition in afoliar application every 4-10 days. In some embodiments, the plant canbe contacted by the composition in a foliar application every 18-24days. In some embodiments, the plant can be contacted by the compositionin a foliar application every 3-7 days. In some embodiments, the plantcan be contacted by the composition in a foliar application every 7-14days. In some embodiments, the plant can be contacted by the compositionin a foliar application every 14-21 days. In some embodiments, the plantcan be contacted by the composition in a foliar application every 21-28days. In some embodiments, the soil or plant can be treated with thecomposition once per planting. In some embodiments, the soil or plantcan be treated with the composition one time every cutting/harvest.

Foliar application(s) of the composition generally begin after the planthas become established, but can begin before establishment, at definedtime period after planting, or at a defined time period after emergenceform the soil in some embodiments. In some embodiments, the plant can befirst contacted by the composition in a foliar application 5-14 daysafter the plant emerges from the soil. In some embodiments, the plantcan be first contacted by the composition in a foliar application 5-7days after the plant emerges from the soil. In some embodiments, theplant can be first contacted by the composition in a foliar application7-10 days after the plant emerges from the soil. In some embodiments,the plant can be first contacted by the composition in a foliarapplication 10-12 days after the plant emerges from the soil. In someembodiments, the plant can be first contacted by the composition in afoliar application 12-14 days after the plant emerges from the soil.

In another non-limiting embodiment, the administration of thecomposition can include contacting the soil in the immediate vicinity ofthe planted seed with an effective amount of the composition. In someembodiments, the liquid composition can be supplied to the soil byinjection into a low volume irrigation system, such as but not limitedto a drip irrigation system supplying water beneath the soil throughperforated conduits or at the soil level by fluid conduits hanging abovethe ground or protruding from the ground. In some embodiments, theliquid composition can be supplied to the soil by a soil drench methodwherein the liquid composition is poured on the soil.

The composition can be diluted to a lower concentration for an effectiveamount in a soil application by mixing a volume of the composition in avolume of water. The percent solids of microalgae sourced componentsresulting in the diluted composition can be calculated by themultiplying the original concentration in the composition by the ratioof the volume of the composition to the volume of water. Alternatively,the grams of microalgae sourced components in the diluted compositioncan be calculated by the multiplying the original grams of microalgaesourced components per 100 mL by the ratio of the volume of thecomposition to the volume of water.

The rate of application of the composition at the desired concentrationcan be expressed as a volume per area. In some embodiments, the rate ofapplication of the liquid composition in a soil application can includea rate in the range of 50-150 gallons/acre. In some embodiments, therate of application of the liquid composition in a soil application caninclude a rate in the range of 75-125 gallons/acre. In some embodiments,the rate of application of the liquid composition in a soil applicationcan include a rate in the range of 50-75 gallons/acre. In someembodiments, the rate of application of the liquid composition in a soilapplication can include a rate in the range of 75-100 gallons/acre. Insome embodiments, the rate of application of the liquid composition in asoil application can include a rate in the range of 100-125gallons/acre. In some embodiments, the rate of application of the liquidcomposition in a soil application can include a rate in the range of125-150 gallons/acre.

In some embodiments, the rate of application of the liquid compositionin a soil application can include a rate in the range of 10-50gallons/acre. In some embodiments, the rate of application of the liquidcomposition in a soil application can include a rate in the range of10-20 gallons/acre. In some embodiments, the rate of application of theliquid composition in a soil application can include a rate in the rangeof 20-30 gallons/acre. In some embodiments, the rate of application ofthe liquid composition in a soil application can include a rate in therange of 30-40 gallons/acre. In some embodiments, the rate ofapplication of the liquid composition in a soil application can includea rate in the range of 40-50 gallons/acre.

In some embodiments, the rate of application of the liquid compositionin a soil application can include a rate in the range of 0.01-10gallons/acre. In some embodiments, the rate of application of the liquidcomposition in a soil application can include a rate in the range of0.01-0.1 gallons/acre. In some embodiments, the rate of application ofthe liquid composition in a soil application can include a rate in therange of 0.1-1.0 gallons/acre. In some embodiments, the rate ofapplication of the liquid composition in a soil application can includea rate in the range of 1-2 gallons/acre. In some embodiments, the rateof application of the liquid composition in a soil application caninclude a rate in the range of 2-3 gallons/acre. In some embodiments,the rate of application of the liquid composition in a soil applicationcan include a rate in the range of 3-4 gallons/acre. In someembodiments, the rate of application of the liquid composition in a soilapplication can include a rate in the range of 4-5 gallons/acre. In someembodiments, the rate of application of the liquid composition in a soilapplication can include a rate in the range of 5-10 gallons/acre.

In some embodiments, the rate of application of the liquid compositionin a soil application can include a rate in the range of 2-20liters/acre. In some embodiments, the rate of application of the liquidcomposition in a soil application can include a rate in the range of3.7-15 liters/acre. In some embodiments, the rate of application of theliquid composition in a soil application can include a rate in the rangeof 2-5 liters/acre. In some embodiments, the rate of application of theliquid composition in a soil application can include a rate in the rangeof 5-10 liters/acre. In some embodiments, the rate of application of theliquid composition in a soil application can include a rate in the rangeof 10-15 liters/acre. In some embodiments, the rate of application ofthe liquid composition in a soil application can include a rate in therange of 15-20 liters/acre.

Prior patent applications containing useful background information andtechnical details are PCT/US2017/053432 titled METHODS OF CULTURINGAURANTIOCHYTRIUM USING ACETATE AS AN ORGANIC CARBON SOURCE, filed onSep. 26, 2017; PCT/US2015/066160, titled MIXOTROPHIC CHLORELLA-BASEDCOMPOSITION, AND METHODS OF ITS PREPARATION AND APPLICATION TO PLANTS,filed on Dec. 15, 2015; and PCT/US2017/037878 and PCT/2017/037880, bothapplications titled MICROALGAE-BASED COMPOSITION, AND METHODS OF ITSPREPARATION AND APPLICATION TO PLANTS, both filed on Jun. 16, 2017. Eachof these applications is incorporated herein by reference in itsentirety.

It will be understood that various modifications may be made to theembodiments disclosed herein. Therefore, the above description shouldnot be construed as limiting, but merely as exemplifications ofpreferred embodiments. Those skilled in the art will envision othermodifications within the scope and spirit of the claims appended hereto.All patents and references cited herein are explicitly incorporated byreference in their entirety.

EXAMPLES

Applying a microalgae composition to plants increases the shelf-lifequality and consumer taste preference of harvested fruits. Shelf-lifequality may include characteristics or metrics such as, but not limitedto, fruit water retention, firmness, and reduction of bruising.Improvement of these factors leads to higher marketability of thefruits. Generally, fruits are stored after harvest at room temperatureor in cold storage (<40° F.) for 5-20 days depending on the trial. Forconsumer preference 80-100 respondents were recruited and polled fortheir preference of treated and untreated strawberries.

Shelf-life metrics described in the Examples below may include: fruitwater-retention; fruit firmness; reduction of bruising; consumerpreference for appearance, overall liking, aroma, texture, and flavor(hedonics); and Christmas tree needle loss and water usage.Water-retention leads to a longer shelf-life due to the fact that thefruit maintains its water content longer post-harvest. Fruit isharvested and weighed immediately and then weighed again after a periodof time specified for each trial. The difference in weight is attributedto the amount of water lost during the storage period. For testingfirmness, a penetrometer is used to measure the force that it takes topenetrate the fruit surface. A firmer fruit indicates longer shelf-life.With respect to bruising, stored fruit is assessed for appearance andbruising is scored as “slight,” “moderate,” “severe,” or “very severe.”Berries with no bruising, slight/light bruising, and moderate bruisingare considered “marketable.” For determining consumer preference forappearance, overall liking, aroma, texture, and flavor (hedonics), asubset of treatments from 3 trials was shipped to a sensory lab forconsumer testing. Reduced needle loss is an indicator of longershelf-life of cut Christmas trees.

Example 1

A trial was conducted on strawberry (var. Camarosa) in Winter Garden,Fla. to evaluate performance of the PHYCOTERRA® Chlorella microalgaecomposition on berry quality after storage and on consumer preference.The trial was transplanted in late October 2016 and harvested throughearly March 2017. All plots were managed according to the local standardpractice (see Study Parameters below).

STUDY PARAMETERS Crop Strawberry (var. Camarosa) Location Winter Garden,FL Transplanting Date Oct. 24, 2016 Pick Frequency Weekly culls when notassessed Bed dimensions 60″ W × 6″ H, 2 rows Planting density 17,424plants/A Drip irrigation 6″ emitters, 0.3″ applied daily Fertilizer20-100 lbs 20-20-20 NPK monthly via drip Pesticide Ridomil, Sevin, Dipeland Captec as needed Soil Type Sandy, non-fumigated Plot Size 12 ftsections of 100 ft bed Replication 4 Product applied 0.5 gal/A via dripat planting then every 2 wks

All plots received the standard fertilization regimen used by the growerfor these crops excluding biostimulants. The microalgae compositionswere added in addition to standard fertilization. Strawberry plants weretransplanted to the field. The first product application was at the timeof transplanting and then every 14 days afterward until harvest via dripirrigation. The untreated control received the same amount of carrierwater as other treatments at the time of each application. Products wereshaken well before application and agitated while in the chemigationtank to prevent solids from settling.

Application rates of the PHYCOTERRA® Chlorella microalgae compositionwere as detailed in Table 1 below. Raw data is included in the tableshown in FIG. 1.

TABLE 1 Treatments Application Treatment Rate Number Product gallon/acreT1 Standard Practice (Untreated) N/A T2 PHYCOTERRA ® composition 0.4 T3PHYCOTERRA ® composition 0.5 T4 PHYCOTERRA ® composition 1 T5PHYCOTERRA ® composition 2 T14 Seaweed-based Commercial Reference 0.5

Berries from one harvest (day 113) were shipped cold overnight to aUniversity lab in New York, stored for 3 days and then assessed forpost-storage quality, particularly appearance. Berries from anadditional harvest (day 128) were shipped to the Sensory EvaluationCenter at a University in New York for consumer testing. Relative tostandard practice alone (UTC), bi-weekly additions of the PHYCOTERRA®Chlorella microalgae composition at 0.5 gal/A improved berry appearanceafter shipping and cold storage. Consumers consistently preferredberries that had come from plots treated with the microalgae compositionover standard practice.

As shown in FIG. 2, berries from a mid-season harvest were assessed forshelf-life quality after being shipped cold to the University lab andstored for 3 days at 34° F. Post-storage marketability was determined byassessing the percent of stored berries with no to slight bruising,compared to those with moderate to severe bruising (unmarketable).Post-storage marketability was significantly improved for berries fromplots treated with the microalgae composition. Referring to FIG. 3,berries from plots receiving the PHYCOTERRA® Chlorella microalgaecomposition were preferred slightly more than those grown using localstandard practice overall and for appearance, aroma, flavor, and texturewhen tested 2 days post-harvest.

In summary, for the Florida trial, the PHYCOTERRA® Chlorella microalgaecomposition reduced the effects of post-harvest shipping and storage onberry quality (e.g. less bruising and improved appearance, aroma,flavor, and texture).

Example 2

A trial was conducted on strawberry (var. Camarosa) in Winter Garden,Fla. to evaluate performance of each of the PHYCOTERRA® Chlorellamicroalgae composition, the Aurantiochytrium acetophilum HS399 extractedbiomass (EB) microalgae composition, and the Greenwater Polyculturemicroalgae composition on strawberry shelf-life and qualitypost-harvest. All plots received the standard fertilization regimen usedby the grower for these crops excluding biostimulants. The Chlorellamicroalgae composition, the Aurantiochytrium acetophilum HS399 extractedbiomass (EB) microalgae composition, and GWP were added in addition tostandard fertilization. Strawberry plants were transplanted to thefield. First application of the microalgae compositions each occurred attime of transplanting and then every 14 days afterward until harvest viadrip irrigation. The UTC received the same amount of carrier water asother treatments at the time of each application. The microalgaecompositions were shaken well before application and agitated while inchemigation tank to prevent solids from settling. All plots were managedaccording to the local standard practice (see Study Parameters below).

STUDY PARAMETERS Crop Strawberry (var. Camarosa) Location Winter Garden,FL Conventional Row Spacing 2 rows/60″ bed with plants every 12″ Plotsize minimum 12 ft section after first 2 ft along 100 ft drip lineTreatment Treatment rows were randomized across field and untreatedbuffer rows were planted on both ends to minimize edge effects One 100ft row, randomized in order throughout field Observations From multiplesubsamples per 12 ft section Replication 14 treatments × 8 replicates =112 treatment plots (12 ft sections) Local Standard ProductionFertility, weed, insect management Standard Management Fungicideapplication. Record disease management measures Practice

Application rates of the PHYCOTERRA® Chlorella microalgae composition,the Aurantiochytrium acetophilum HS399 extracted biomass (EB) microalgaecomposition, and the Greenwater Polyculture microalgae composition wereas detailed in Table 2 below.

TABLE 2 Treatments Application Treatment Rate Number Product gallon/acreT1 Standard Practice (Untreated) N/A T2 PHYCOTERRA ® composition 0.4 T3PHYCOTERRA ® composition 0.5 T4 PHYCOTERRA ® composition 1 T5PHYCOTERRA ® composition 2 T6 HS399 Extracted Biomass (EB) 0.4 T7 HS399Extracted Biomass (EB) 0.5 T8 HS399 Extracted Biomass (EB) 1 T9 HS399Extracted Biomass (EB) 2 T10 Green Water Polyculture 0.4 T11 Green WaterPolyculture 0.5 T12 Green Water Polyculture 1 T13 Green WaterPolyculture 2 T14 Seaweed-based Commercial Reference 0.5

Marketable berries from all replicates were harvested. Replicate 2 lbclamshells were kept for an on-site storage assessment or shippedovernight to a university lab in NY for an additional storageassessment. On-site, clamshells were placed at 38° F. for 4-5 days,followed by 24 hrs at ambient temperature. Berry quality was assessedafter the storage period, using sub-sampling of individual berries whenappropriate for the following characteristics: sweetness (% brix),firmness, post-harvest disease & decay. Once the second batch ofreplicates was received by the University, they were stored for 4 daysat 34° F. and then assessed for post-storage quality.

A second harvest was conducted for a sensory panel. Upon harvest, 100berries of similar size and ripeness from the standard practice and 2-3additional treatments were packed into clamshells and shipped to aUniversity sensory lab for sensory panel evaluation. Clamshells werewrapped in bubble wrap and shipped on blue ice.

As shown in FIGS. 4-5, sixteen weeks after planting, strawberries wereharvested and either kept in cold storage onsite or shipped overnight tothe university lab where they were kept in cold storage. After a periodof 4 days in storage, at both sites, berries were assessed for bruiseseverity and marketability. With few exceptions, all treatments improvedmarketability compared to standard practice by >20%. After shipping andstorage, almost all treatments also improved marketability over theseaweed-based commercial reference product. Similar patterns wereobserved for the reduction of severe and moderate bruising. Aftershipping and storage, the PHYCOTERRA® Chlorella microalgae compositionand the Aurantiochytrium acetophilum HS399 extracted biomass (EB)microalgae composition showed the most advantage.

In summary, for the Florida trial, the microalgae composition reducedthe effects of post-harvest shipping and storage on berry quality (e.g.less bruising and improved appearance, aroma, flavor, and texture).Specifically, for plots receiving the PHYCOTERRA® Chlorella microalgaecomposition, marketability due to the bruising level of the fruits wasimproved by between 16-98% over the UTC and were improved by between15-98% over the seaweed commercial reference. Furthermore, for plotsreceiving the PHYCOTERRA® Chlorella microalgae composition, the amountof severe to moderate bruising of the fruits was decreased by between11-88% compared to the UTC and was decreased by between 5-35% comparedto the seaweed commercial reference.

For plots receiving the Aurantiochytrium acetophilum HS399 extractedbiomass (EB) microalgae composition, marketability due to the bruisinglevel of the fruits was improved by between 24-116% over the UTC andwere improved by between 1-115% over the seaweed commercial reference.Furthermore, for plots receiving the Aurantiochytrium acetophilum HS399extracted biomass (EB), the amount of severe to moderate bruising of thefruits was decreased by between 11-100% compared to the UTC and wasdecreased by between 20-37% compared to the seaweed commercialreference.

For plots receiving the GWP microalgae composition, marketability due tothe bruising level of the fruits was improved by between 3-72% over theUTC and were improved by between 2-72% over the seaweed commercialreference. Furthermore, for plots receiving the GWP microalgaecomposition, the amount of severe to moderate bruising of the fruits wasdecreased by between 17-100% compared to the UTC and was decreased bybetween 1-26% compared to the seaweed commercial reference.

Nineteen weeks after planting, strawberries were harvested and shippedovernight to a university sensory lab where they were kept at ambientconditions. The next day they were assessed by 74 respondents forvarious hedonics attributes. All treatments were tested at the 0.5gallon/acre rate. Referring to FIG. 6, for every attribute, thePHYCOTERRA® Chlorella microalgae composition and the seaweed commercialreference were preferred over standard practice. The PHYCOTERRA®Chlorella microalgae composition was especially preferred over mosttreatments for aroma and texture liking. The Aurantiochytriumacetophilum HS399 extracted biomass (EB) microalgae composition wasespecially preferred for flavor liking. Greenwater Polyculture (GWP)tracked with other treatments in terms of advantages over standardpractice.

Example 3

A trial was conducted on strawberry (var. Portola) in Guadalupe Valley,Calif. to evaluate performance of various microalgae compositions onstrawberry growth, yield, and post-harvest berry quality; specifically,the PHYCOTERRA® Chlorella microalgae composition, the OMRI certifiedTERRENE® Chlorella pasteurized at 90° C. microalgae composition, theAurantiochytrium acetophilum HS399 whole biomass (WB) microalgaecomposition, the Aurantiochytrium acetophilum HS399 washed whole biomass(WB washed) microalgae composition, and the combination 25% Chlorella:75% HS399 whole biomass (WB) microalgae composition. All plots receivedstandard local fertilization regimen used by the grower for this cropexcluding biostimulants. Each of the microalgae compositions were addedin addition to standard fertilization. Strawberry plants weretransplanted to the field in early June 2017, according to localcommercial practice. The first product application was via dripirrigation at the time of transplanting and then every 14 days afterwarduntil harvest. The untreated control received the same amount of carrierwater as other treatments at the time of each product application. Themicroalgae compositions were shaken well before application andagitated, if possible, while in the chemigation tank to prevent solidsfrom settling. Berries were harvested according to local commercialschedule. All plots were managed according to the local standardpractice (see Study Parameters below). Raw data is shown in Table 3below.

STUDY PARAMETERS Crop Strawberry (var. Portola) Location GuadalupeValley, CA Conventional Row Spacing 40″ furrow spacing with 24″ wide bedspacing, and plants on plant lines 12″ apart and plant lines 12″ apartHarvest Schedule As frequently as standard local grower practice withestimated 12-16 picks Fumigation Schedule Early May, 32 gal/a PicChlor60Plot size minimum 1 double-line bed 45 ft length per plot with 80+plants per plot Trial Design Randomized Complete Block ObservationsTaken from 70 plants inside 3 ft buffer zone of each plot endReplication 6 replicate plots for each treatment Local StandardProduction Fertility, weed, insect management, etc. Standard ManagementFungicide application. Record disease management measures PracticeFungicides will be applied weekly when flowers and fruit are present

TABLE 3 Raw Data % % Advantage over Day 5% % Advantage over Day 7% %Advantage over Holding Rate Weight Standard Comm. brusing Standard Comm.bruising Standard Comm. Test Treatment (gal/A) loss Practice Ref. levelPractice Ref. level Practice Ref. 1 Standard Practice 2.13 1.28 1.63Comm. Ref. 0.50 1.88 −12% 1.22 −5% 1.40 −14% PhycoTerra 0.25 1.87 −12%−1% 0.93 −27% −23% 1.43 −12% 2% PhycoTerra 0.50 1.77 −17% −6% 1.10 −14%−10% 1.47 −10% 5% Terrene90 0.25 1.78 −16% −5% 1.02 −21% −16% 1.43 −12%2% Terrene90 0.50 1.85 −13% −2% 1.12 −13% −8% 1.48 −9% 6% HS399 WB 0.251.90 −11% 1% 1.07 −17% −12% 1.35 −17% −4% HS399 WB 0.50 1.77 −17% −6%0.98 −23% −19% 1.42 −13% 1% HS399 WB washed 0.25 1.83 −14% −3% 1.17 −9%−4% 1.45 −11% 4% HS399 WB washed 0.50 1.78 −16% −5% 0.95 −26% −22% 1.25−23% −11% Combo 399WB 0.25 1.78 −16% −5% 0.93 −27% −23% 1.30 −20% −7%Combo 399WB 0.50 1.83 −14% −3% 1.15 −10% −5% 1.53 −6% 10%

Application rates of the PHYCOTERRA® Chlorella microalgae composition,the OMRI certified TERRENE® Chlorella microalgae composition pasteurizedat 90° C., the Aurantiochytrium acetophilum HS399 whole biomass (WB)microalgae composition, the Aurantiochytrium acetophilum HS399 washedwhole biomass (WB washed) microalgae composition, and the combination25% Chlorella: 75% Aurantiochytrium acetophilum HS399 whole biomass (WB)microalgae composition were as detailed in Table 4 below.

TABLE 4 Treatments Application Treatment Rate Number Product gallon/acreT1 Untreated control (UTC/standard practice) N/A T2 Seaweed CommercialReference 0.5 T3 PHYCOTERRA ® Chlorella composition 0.25 T4 PHYCOTERRA ®Chlorella composition 0.5 T5 HS399 Whole Biomass (WB) washed 0.25 T6HS399 Whole Biomass (WB) washed 0.5 T7 HS399 Whole Biomass (WB) 0.25 T8HS399 Whole Biomass (WB) 0.5 T9 TERRENE ® Chlorella composition 0.25pasteurized at 90° C. T10 TERRENE ® Chlorella composition 0.5pasteurized at 90° C. T11 25% Chlorella: 75% HS399 WB 0.25 T12 25%Chlorella: 75% HS399 WB 0.5

Fifteen weeks after transplanting, berries were harvested and stored incold storage for 5 or 7 days and bruising severity on the stored berrieswas compared between treatments. As shown in FIG. 7, berries frommicroalgae-treated plants had a lower degree of bruising (5-27%reduction) than from plants grown using standard practice. Specifically,for berries treated with the PHYCOTERRA® Chlorella composition, bruisingwas decreased by between 14-27% compared to the UTC and bruising wasdecreased by between 10-23% compared to the seaweed commercialreference. For berries treated with the TERRENE® Chlorella microalgaecomposition pasteurized at 90° C., bruising was decreased by between13-21% compared to the UTC and bruising was decreased by between 8-16%compared to the seaweed commercial reference. For berries treated withthe HS399 Whole Biomass (WB) microalgae composition, bruising wasdecreased by between 17-23% compared to the UTC and bruising wasdecreased by between 12-19% compared to the seaweed commercialreference. For berries treated with the HS399 Whole Biomass (WB) washedmicroalgae composition, bruising was decreased by between 9-26% comparedto the UTC and bruising was decreased by between 4-22% compared to theseaweed commercial reference. For berries treated with the combination25% Chlorella: 75% HS399 WB microalgae combination, bruising wasdecreased by between 10-27% compared to the UTC and bruising wasdecreased by between 5-23% compared to the seaweed commercial reference.

As shown in FIG. 8, berries from microalgae-treated plants had a lowerdegree of bruising (6-23% reduction) than from plants grown usingstandard practice. Specifically, for berries treated with thePHYCOTERRA® Chlorella composition, bruising was decreased by between10-12% compared to the UTC but bruising was not decreased compared tothe seaweed commercial reference. For berries treated with the TERRENE®Chlorella microalgae composition pasteurized at 90° C., bruising wasdecreased by between 9-12% compared to the UTC but bruising was notdecreased compared to the seaweed commercial reference. For berriestreated with the HS399 Whole Biomass (WB) microalgae composition,bruising was decreased by between 13-17% compared to the UTC. Forberries treated with the HS399 Whole Biomass (WB) washed microalgaecomposition, bruising was decreased by between 11-23% compared to theUTC. For berries treated with the combination 25% Chlorella: 75% HS399WB microalgae combination, bruising was decreased by between 6-20%compared to the UTC.

Example 4

For the treatments referred to in this Example as CommercialReference+TERRENE® pasteurized at 65° C., the commercial reference wasapplied first to the soil at a rate of 20 gal/acre. The TERRENE®pasteurized at 65° C. microalgae composition was then added on top viadrip irrigation. The commercial reference was only applied 4 times perseason, whereas the TERRENE® pasteurized at 65° C. microalgaecomposition was applied every 14 days until harvest.

For the treatments referred to in this Example as CommercialReference+TERRENE® pasteurized at 90° C., the commercial reference wasapplied first to the soil at a rate of 20 gal/acre. The TERRENE®pasteurized at 90° C. microalgae composition was then added on top viadrip irrigation. The commercial reference was only applied 4 times perseason, whereas the TERRENE® pasteurized at 90° C. microalgaecomposition was applied every 14 days until harvest.

A trial was conducted on strawberry (var. Portola—Organic) in SantaMaria, Calif. to evaluate performance of various microalgae compositionson strawberry growth, yield, and post-harvest berry quality,particularly the OMRI certified TERRENE® Chlorella pasteurized at 65° C.microalgae composition, the OMRI certified TERRENE® Chlorellapasteurized at 90° C. microalgae composition, the combination OMRIcertified TERRENE® Chlorella pasteurized at 65° C. microalgaecomposition: microbial based commercial reference product microalgaecomposition, and the combination OMRI certified TERRENE® Chlorellapasteurized at 90° C. microalgae composition: microbial based commercialreference product microalgae composition. All plots received standardlocal fertigation practice, including NEPTUNE'S HARVEST fertilizer andNFORCE fertilizer. A control was added with standard local fertigationpractice plus 4 applications of a microbial-based commercial referenceproduct that is standard to this location. Treatments included twoversions of an OMRI certified Chlorella microalgae composition thatdiffer by pasteurization temperature (OMRI certified TERRENE® Chlorellapasteurized at 65° C. microalgae composition and OMRI certified TERRENE®Chlorella pasteurized at 90° C. microalgae composition), each testedalone and each tested in combination with the microbial-based commercialreference. Strawberry plants (frigo) were transplanted to the field inJune 2017, according to local commercial practice. The first productapplication was via drip irrigation at the time of transplanting andthen every 14 days afterward through to final harvest. The untreatedcontrol received the same amount of carrier water as other treatments atthe time of each product application. The microalgae compositions wereshaken well before application and agitated while in the chemigationtank in order to prevent solids from settling. Berries were harvestedaccording to local commercial schedule (twice per week during fruitingseason). The timing of the commercial reference applications was once atthe time of planting (6/20), once 14-21 days after planting (7/5), oncein late July/early August (7/31) and the last in early September (9/11).All plots were managed according to the local standard practice (seeStudy Parameters below).

STUDY PARAMETERS Crop Strawberry (var. Portola) Location Santa Maria, CAConventional Row Spacing Wide 4-row beds, 64-inches center-to-center;plants spaced 14 inches apart in each of the four rows Harvest ScheduleAs frequently as standard local grower practice with estimated 32 picksFumigation Schedule None (Organic) Plot size minimum 1 four-row bed25-30 ft length per plot with 80+ plants per plot. Plots will be locatedaway from any field edges with 1-2 commercial buffer beds in betweenTrial Design Randomized complete block Observations Yield data takenfrom 40 inside plants, outside 40 combined with inside 40 forpost-harvest assessments Replication 6 replicate plots for eachtreatment and untreated control Local Standard Production Fertility,weed, insect management, etc. Standard Management Standard managementpractices for organic production. Practice Record disease managementmeasures

Application rates of the OMRI certified TERRENE® Chlorella pasteurizedat 65° C. microalgae composition, the OMRI certified TERRENE® Chlorellapasteurized at 90° C. microalgae composition treatment, the combinationCommercial Reference+OMRI certified TERRENE® Chlorella pasteurized at65° C. microalgae composition treatment, and the combination CommercialReference+OMRI certified TERRENE® Chlorella pasteurized at 90° C.microalgae composition treatment were as detailed in Table 5 below. Rawdata is included in the table shown in FIG. 9.

TABLE 5 Treatments Application Treatment Rate Number Product gallon/acreT1 Standard practice only (UTC) Water T2 Commercial reference (NoTERRENE ®) 20 T3 Commercial Reference + TERRENE ® 0.5 pasteurized at 65°C. T4 Commercial Reference + TERRENE ® 0.5 pasteurized at 90° C. T5TERRENE ® pasteurized at 65° C. 0.25 T6 TERRENE ® pasteurized at 90° C.0.25 T7 TERRENE ® pasteurized at 65° C. 0.5 T8 TERRENE ® pasteurized at90° C. 0.5

At 11, 14 and 18 weeks after transplanting, berries were harvested andstored in cold storage for 6 days and bruising was assessed at the endof this period. Referring to FIG. 10, marketability (% of berries withmild to moderate bruising) is shown for all 3 assessments performed onSanta Maria berries. In all 3 assessments, the OMRI certified TERRENE®Chlorella pasteurized at 90° C. microalgae composition (0.5 gal/A)showed an increase in marketability due to reduced bruising compared tostandard practice (5-9%). The microbial-based Commercial Reference+OMRIcertified TERRENE® Chlorella pasteurized at 65° C. microalgaecomposition (0.5 gal/A; 11-40% increase in marketability compared toUTC), the OMRI certified TERRENE® Chlorella pasteurized at 65° C.microalgae composition alone (0.25 gal/A; 4-8% increase in marketabilitycompared to UTC), and the OMRI certified TERRENE® Chlorella pasteurizedat 90° C. microalgae composition alone (0.25 gal/A, 2-9% increase inmarketability compared to UTC) all showed an advantage over standardpractice in 2 of 3 assessments. Compared to the commercial referencealone, the OMRI certified TERRENE® Chlorella pasteurized at 65° C.microalgae composition and the OMRI certified TERRENE® Chlorellapasteurized at 90° C. microalgae composition showed an advantageincreasing marketability in 2 of 3 assessments (5-30% over thecommercial reference).

For berries treated with the OMRI certified TERRENE® Chlorellapasteurized at 65° C. microalgae composition alone, marketability due tobruising level was increased by between 4-28% over the UTC and wasincreased by between 5-26% over the commercial reference. For berriestreated with the OMRI certified TERRENE® Chlorella pasteurized at 90° C.microalgae composition alone, marketability due to bruising level wasincreased by between 2-9% over the UTC and was increased by between15-33% over the commercial reference. For berries treated with thecombination Commercial Reference+OMRI certified TERRENE® Chlorellapasteurized at 65° C. microalgae composition, marketability due tobruising was increased by between 11-40% over the UTC and increased bybetween 9-21% over the commercial reference alone.

The percent of berries with severe bruising is also shown in FIG. 11.Compared to standard practice, the OMRI certified TERRENE® Chlorellapasteurized at 65° C. microalgae composition (0.25 gal/A) and thecombination of the Commercial Reference+OMRI certified TERRENE®Chlorella pasteurized at 90° C. microalgae composition reduced severebruising (10-30%) in 2 of 3 assessments. Compared to the commercialreference, multiple treatments showed a benefit.

For berries treated with the OMRI certified TERRENE® Chlorellapasteurized at 65° C. microalgae composition alone, severe bruising wasdecreased by between 10-19% compared to the UTC and was decreased bybetween 24-48% compared to the commercial reference. For berries treatedwith the OMRI certified TERRENE® Chlorella pasteurized at 90° C.microalgae composition alone, severe bruising was decreased by between3-22% compared to the UTC and was decreased by between 18-50% comparedto the commercial reference. For berries treated with the combinationCommercial Reference+OMRI certified TERRENE® Chlorella pasteurized at65° C. microalgae composition, severe bruising was decreased by between3-68% compared to the UTC. For berries treated with the combinationCommercial Reference+OMRI certified TERRENE® Chlorella pasteurized at90° C. microalgae composition, severe bruising was decreased by 10-21%compared to the UTC and was decreased by between 24-55% compared to thecommercial reference.

For the consumer preference testing, one harvest was performed for eachtrial. The first occurred on or about Oct. 11, 2017. Marketable berriesfrom 6 replicates each of 6 treatments were harvested and packaged intoclamshells. These were delivered to the University sensory lab the sameday. The second harvest occurred on or about Nov. 2, 2017. Marketableberries from 6 replicates each of 5 treatments were harvested andpackaged into clamshells. Once shipment was received, a consumer tastepanel was conducted. Berries were washed and prepared into halves forapproximately 100 consumer volunteers who received one half of 5-6berries (UTC and 4-5 treatments). Berries were scored for appearance andflavor. It should be noted that for the consumer sensory panel testing,the treatments were as shown below in Table 6.

TABLE 6 Treatments Application Treatment Rate Number Product gallon/acreT1 Standard practice only (UTC) Water T2 Commercial reference (NoTERRENE ®) 20 T3 Commercial Reference + TERRENE ® 0.5 pasteurized at 65°C. T5 TERRENE ® pasteurized at 65° C. 0.25 T7 TERRENE ® pasteurized at65° C. 0.5

Twelve weeks after transplanting, berries were harvested and transportedto a University sensory lab where they were kept overnight at 4° C. Thenext day they were assessed by 102 respondents for various likingattributes. Referring to FIG. 12, the OMRI certified TERRENE® Chlorellapasteurized at 65° C. microalgae composition applied at ½ gal/A waspreferred over standard practice for overall liking and flavor likingand generally performed similarly to the microbial-based commercialreference. The commercial reference was preferred over standard practicefor all attributes.

Example 5

A trial was conducted on strawberry (var. Portola) in Oxnard, Calif. toevaluate performance of various microalgae compositions on strawberrygrowth, yield, and post-harvest berry quality; particularly, thePHYCOTERRA® Chlorella microalgae composition, the OMRI certifiedTERRENE® Chlorella pasteurized at 65° C. microalgae composition, theAurantiochytrium acetophilum HS399 whole biomass (WB) microalgaecomposition, the Aurantiochytrium acetophilum HS399 extracted biomass(EB) microalgae composition, and the combination 25% Chlorella: 75%HS399 whole biomass (WB) microalgae composition. All plots receivestandard local fertilization regimen used by the grower for this crop,excluding biostimulants. The microalgae compositions were added inaddition to standard fertilization. Strawberry plants were transplantedto the field in July 2017, according to local commercial practice. Thefirst product application was via drip irrigation at the time oftransplanting and then every 14 days afterward through to final harvest.The untreated control received the same amount of carrier water as othertreatments at the time of each product application. The microalgaecompositions were shaken well before application and agitated, ifpossible, while in the chemigation tank to prevent solids from settling.Berries were harvested according to local commercial schedule (twice perweek during the fruiting season). All plots were managed according tothe local standard practice (see Study Parameters below).

STUDY PARAMETERS Crop Strawberry (var. Portola) Location Oxnard, CAConventional Row Spacing Wide 4-row beds, 64-inches center-to-center;plants spaced 14 inches apart in each of the four rows Harvest ScheduleAs frequently as standard local grower practice with estimated 24 picksFumigation Schedule Local practice (recorded) - timing will be in JunePlot size minimum 1 four-row bed 25 ft length per plot with 80+ plantsper plot. Plots will be located away from any field edges with 1-2commercial buffer beds in between Trial Design Randomized complete blockObservations Yield data taken from 40 inside plants, outside 40 combinedwith inside 40 for post-harvest assessments Replication 6 replicateplots for each treatment and untreated control Local Standard ProductionFertility, weed, insect management, etc Standard Management Standardmanagement practices, including fungicide Practice application. Recorddisease management measures. Fungicides will be applied as necessary (bygrower) when flowers and fruit are present

Application rates of the PHYCOTERRA® Chlorella microalgae composition,the Aurantiochytrium acetophilum HS399 extracted biomass (EB) microalgaecomposition, the Aurantiochytrium acetophilum HS399 whole biomass (WB)microalgae composition, the OMRI certified TERRENE® Chlorellapasteurized at 65° C. microalgae composition, and the combination 25%Chlorella: 75% HS399 whole biomass (WB) microalgae composition were asdetailed in Table 7 below. Raw data is included in the table shown inFIGS. 13-14.

TABLE 7 Treatments Application Treatment Rate Number Product gallon/acreT1 Untreated control (UTC/standard practice) Water T2 Seaweed CommercialReference 0.5 T3 PHYCOTERRA ® Chlorella composition 0.25 T4 PHYCOTERRA ®Chlorella composition 0.5 T5 HS399 Extracted Biomass (EB) 0.25 T6 HS399Extracted Biomass (EB) 0.5 T7 HS399 Whole Biomass (WB) 0.25 T8 HS399Whole Biomass (WB) 0.5 T9 TERRENE ® pasteurized at 65° C. 0.25 T10TERRENE ® pasteurized at 65° C. 0.5 T11 25% Chlorella: 75% HS399 WB 0.25T12 25% Chlorella: 75% HS399 WB 0.5

At 11, 15, 19, 21 and 26 weeks after transplanting, berries wereharvested and stored in cold storage for 6-10 days and bruising wasassessed at the end of this period. Marketability (% of berries withmild to moderate bruising) is shown in FIG. 15 for all 4 assessmentsperformed on Oxnard strawberries. Compared to standard practice,improvements were only observed for the final two assessments (lateseason). The OMRI certified TERRENE® Chlorella pasteurized at 65° C.microalgae composition (6-18% increase) and the Aurantiochytriumacetophilum HS399 extracted biomass (EB) microalgae composition (3-20%increase) showed increased marketability due to bruising reduction inboth assessments compared to the UTC. Improvements over the seaweedcommercial reference were only observed in the first and last assessment(early and late season). All microalgae compositions showed an advantageover the commercial reference for the first assessment (2-20%) and forthe late season final assessment of PHYCOTERRA® Chlorella microalgaecomposition (0.25 gal/A), the OMRI certified TERRENE® Chlorellapasteurized at 65° C. microalgae composition (0.25 gal/A), and bothrates of Aurantiochytrium acetophilum HS399 extracted biomass (EB)microalgae composition (6-11%). As shown in FIG. 16, the percent ofberries with severe bruising was especially reduced during late seasonby multiple products (10-60%).

For berries treated with the PHYCOTERRA® Chlorella microalgaecomposition, marketability due to bruising level increased by between6-19% over the UTC and was increased by between 3-6% over the commercialreference. For berries treated with the OMRI certified TERRENE®Chlorella pasteurized at 65° C. microalgae composition, marketabilitydue to bruising level increased by between 6-18% over the UTC and wasincreased by between 6-18% over the commercial reference. For berriestreated with the Aurantiochytrium acetophilum HS399 extracted biomass(EB) microalgae composition, marketability due to bruising levelincreased by between 3-24% over the UTC and was increased by between2-15% over the commercial reference. For berries treated with theAurantiochytrium acetophilum HS399 whole biomass (WB) microalgaecomposition, marketability due to bruising level increased by between9-17% over the commercial reference. For berries treated with thecombination 25% Chlorella: 75% HS399 whole biomass (WB) microalgaecomposition marketability due to bruising level increased by between4-20% over the commercial reference.

For berries treated with the PHYCOTERRA® Chlorella microalgaecomposition, severe bruising was decreased by between 10-54% compared tothe UTC and was decreased by between 3-28% compared to the commercialreference. For berries treated with the OMRI certified TERRENE®Chlorella pasteurized at 65° C. microalgae composition, severe bruisingwas decreased by between 2-61% compared to the UTC and was decreased bybetween 9-22% compared to the commercial reference. For berries treatedwith the Aurantiochytrium acetophilum HS399 extracted biomass (EB)microalgae composition, severe bruising was decreased by between 4-58%compared to the UTC and was decreased by between 18-34% compared to thecommercial reference. For berries treated with the Aurantiochytriumacetophilum HS399 whole biomass (WB) microalgae composition, severebruising was decreased by between 2-20% compared to the UTC and wasdecreased by between 17-22% compared to the commercial reference. Forberries treated with the combination 25% Chlorella: 75% HS399 wholebiomass (WB) microalgae composition, severe bruising was decreased bybetween 4-15% compared to the UTC and was decreased by between 5-33%compared to the commercial reference.

For the consumer preference testing, one harvest was performed for eachtrial. The first occurred on or about Oct. 11, 2017. Marketable berriesfrom 6 replicates each of 6 treatments were harvested and packaged intoclamshells. These were delivered to the University sensory lab the sameday. The second harvest occurred on or about Nov. 2, 2017. Marketableberries from 6 replicates each of 5 treatments were harvested andpackaged into clamshells. Once shipment was received, a consumer tastepanel was conducted. Berries were washed and prepared into halves forapproximately 100 consumer volunteers who received one half of 5-6berries (UTC and 4-5 treatments). Berries were scored for appearance andflavor. It should be noted that for the consumer sensory panel testing,the treatments were as shown below in Table 8.

TABLE 8 Treatments Application Treatment Rate Number Product gallon/acreT1 Standard practice only (UTC) Water T2 Seaweed Commercial Reference0.5 T4 PHYCOTERRA ® Chlorella composition 0.5 T6 HS399 Extracted Biomass(EB) 0.5 T8 HS399 Whole Biomass (WB) 0.5 T10 TERRENE ® pasteurized at65° C. 0.5

Sixteen weeks after transplanting, berries were harvested andtransported to a university sensory lab where they were kept overnightat 4° C. The next day they were assessed by 100 respondents for variousliking attributes. All treatments were tested at the ½ gallon/A rate. Asshown in FIG. 17, the Aurantiochytrium acetophilum HS399 whole biomass(WB) microalgae composition was preferred over standard practice and theseaweed-based commercial reference for all attributes. This treatmentparticularly stood out for overall liking, aroma and flavor. ThePHYCOTERRA® Chlorella composition was also preferred over standardpractice for all attributes but not to the same degree as theAurantiochytrium acetophilum HS399 whole biomass (WB) microalgaecomposition. The OMRI certified TERRENE® Chlorella pasteurized at 65° C.microalgae composition was only preferred over standard practice forappearance and was preferred less for overall liking, aroma and flavor.

All references, including publications, patent applications, andpatents, cited herein are hereby incorporated by reference in theirentirety and to the same extent as if each reference were individuallyand specifically indicated to be incorporated by reference and were setforth in its entirety herein (to the maximum extent permitted by law),regardless of any separately provided incorporation of particulardocuments made elsewhere herein.

The use of the terms “a” and “an” and “the” and similar referents in thecontext of describing the invention are to be construed to cover boththe singular and the plural, unless otherwise indicated herein orclearly contradicted by context.

Unless otherwise stated, all exact values provided herein arerepresentative of corresponding approximate values (e.g., all exactexemplary values provided with respect to a particular factor ormeasurement can be considered to also provide a correspondingapproximate measurement, modified by “about,” where appropriate). Allprovided ranges of values are intended to include the end points of theranges, as well as values between the end points.

The description herein of any aspect or embodiment of the inventionusing terms such as “comprising”, “having,” “including,” or “containing”with reference to an element or elements is intended to provide supportfor a similar aspect or embodiment of the invention that “consists of”,“consists essentially of”, or “substantially comprises” that particularelement or elements, unless otherwise stated or clearly contradicted bycontext (e.g., a composition described herein as comprising a particularelement should be understood as also describing a composition consistingof that element, unless otherwise stated or clearly contradicted bycontext).

All headings and sub-headings are used herein for convenience only andshould not be construed as limiting the invention in any way.

The use of any and all examples, or exemplary language (e.g., “such as”)provided herein, is intended merely to better illuminate the inventionand does not pose a limitation on the scope of the invention unlessotherwise claimed. No language in the specification should be construedas indicating any non-claimed element as essential to the practice ofthe invention.

The citation and incorporation of patent documents herein is done forconvenience only and does not reflect any view of the validity,patentability, and/or enforceability of such patent documents.

This invention includes all modifications and equivalents of the subjectmatter recited in the claims and/or aspects appended hereto as permittedby applicable law.

What is claimed is:
 1. A method of decreasing bruising of fruit of afruiting plant comprising the step of administering to the fruitingplant, seedling, or seed a liquid composition treatment comprising aculture of microalgae, the microalgae comprising at least one ofpasteurized Chlorella cells and pasteurized Aurantiochytrium acetophilumHS399 cells in an amount effective to decrease bruising in the fruit ofa population of such fruiting plants compared to a substantiallyidentical population of untreated fruiting plants.
 2. The method ofclaim 1 wherein administering comprises contacting soil in the immediatevicinity of the fruiting plant, seedling, or seed with an effectiveamount of the liquid composition treatment.
 3. The method of claim 2wherein the liquid composition is administered at a rate in the range of0.25-2 gallons/acre.
 4. The method of claim 3 wherein the liquidcomposition comprises between 100 g-800 g per acre of at least one ofpasteurized Chlorella cells and pasteurized Aurantiochytrium acetophilumHS399 cells.
 5. The method of claim 1 wherein the liquid compositiontreatment further comprises phosphoric acid and potassium sorbate. 6.The method of claim 1 wherein the liquid composition treatment furthercomprises citric acid.
 7. The method of claim 1 wherein the pasteurizedChlorella cells are pasteurized at a temperature in the range of 65°C.-90° C. and the pasteurized Aurantiochytrium acetophilum HS399 cellsare pasteurized at a temperature in the range of 65° C.-75° C.
 8. Themethod of claim 1 wherein the at least one of pasteurized Chlorellacells and pasteurized Aurantiochytrium acetophilum HS399 cells arepasteurized for between 90-150 minutes.
 9. The method of claim 1 whereinthe microalgae comprises only Chlorella cells and the liquid compositionis applied in an effective amount to decrease bruising in fruit of thefruiting plant by between 11-88% compared to the substantially identicalpopulation of untreated fruiting plants.
 10. The method of claim 1wherein the microalgae comprises only Aurantiochytrium cells and theliquid composition is applied in an amount effective to decreasebruising in fruit of the fruiting plant by between 2-100% compared tothe substantially identical population of untreated fruiting plants. 11.The method of claim 1 wherein the microalgae comprises Chlorella cellsand Aurantiochytrium acetophilum HS399 cells in an amount effective todecrease bruising in fruit of the fruiting plant by between 3-68%compared to the substantially identical population of untreated fruitingplants.
 12. The method of claim 11 wherein the liquid compositioncomprises pasteurized Chlorella cells and pasteurized Aurantiochytriumacetophilum HS399 cells in a ratio of 25:75.
 13. The method of claim 1wherein the Aurantiochytrium acetophilum HS399 cells have been subjectedto an extraction process to remove oils from the Aurantiochytriumacetophilum HS399 cells.
 14. A method of decreasing bruising of fruit ofa fruiting plant comprising the step of administering to the fruitingplant, seedling, or seed a liquid composition treatment comprising aculture of microalgae, the microalgae comprising at least one ofpasteurized Chlorella cells and pasteurized Aurantiochytrium acetophilumHS399 cells in an effective amount to decrease bruising in the fruit ofa population of fruiting plants by 2-100% compared to fruit of asubstantially identical population of untreated fruiting plants, whereinadministering comprising contacting soil in the immediate vicinity ofthe fruiting plant, seedling, or seed with an effective amount of theliquid composition treatment by drip irrigation.
 15. The method of claim14 wherein the liquid composition comprises pasteurized Chlorella cellsand pasteurized Aurantiochytrium acetophilum HS399 cells in a ratio of25:75.
 16. The method of claim 14 wherein the Aurantiochytriumacetophilum HS399 cells have been subjected to an extraction process toremove oils from the Aurantiochytrium acetophilum HS399 cells.
 17. Themethod of claim 14 wherein the liquid composition comprises between 100g-800 g per acre of at least one of pasteurized Chlorella cells andpasteurized Aurantiochytrium acetophilum HS399 cells.
 18. A method ofdecreasing bruising of fruit of a fruiting plant comprising the stepsof: providing a liquid composition treatment comprising a culture ofmicroalgae, the microalgae comprising at least one of pasteurizedChlorella cells and pasteurized Aurantiochytrium acetophilum HS399cells; diluting the liquid composition treatment to contain between 100g-800 g per acre of the at least one of pasteurized Chlorella cells andpasteurized Aurantiochytrium acetophilum HS399 cells; and administeringthe liquid composition treatment to a fruiting plant, seedling, or seedin an effective amount to decrease bruising in the fruit of a populationof such fruiting plants compared to a substantially identical populationof untreated fruiting plants.
 19. The method of claim 18, wherein theliquid composition is administered by contacting soil in the immediatevicinity of the fruiting plant, seedling, or seed with an effectiveamount of the liquid composition treatment.
 20. The method of claim 18wherein bruising of the fruit is decreased by 2-100% compared to fruitof a substantially identical population of untreated fruiting plants.